Platinum complexes and uses thereof

ABSTRACT

The disclosure generally relates to compositions comprising therapeutic agent complexes and to methods of making and using the compositions. In particular embodiments, the disclosure provides compositions comprising platinum-based drug complexes and to methods of making and using these compositions.

BACKGROUND

Platinum-based compounds are among the most widely used drug classes incancer therapy. Almost half of the chemotherapy regimens contain aplatinum drug. The widespread use of platinum agents in the treatment ofcancer began with the discovery of the antineoplastic activity ofcisplatin in the 1960s. Despite the pervasiveness of platinum drugs incancer treatment regimens, broader application of platinum-based drugssuch as cisplatin and oxaliplatin is ultimately limited by their lowsolubility, short half-life, and the substantial risk of severetoxicities, including neurotoxicity. Improvement of drug targeting anddelivery systems are effective approaches to mitigate thesedisadvantages.

There is, therefore, a great need for developing platinum-based drugtargeting and delivery systems that are capable preferentiallydelivering cytotoxic platinum drug-based payloads that have widertherapeutic indices and more favorable toxicity profiles than currentlyadministered.

The provided compositions preferentially deliver therapeutic agentpayloads, such as platinum-based drugs to hyperproliferative cellsincluding cancer cells and provide significant improvements in efficacyand safety over conventional platinum therapy-based treatment regimens.

BRIEF SUMMARY

The disclosure generally relates to compositions comprising therapeuticagent complexes and to methods of making and using the compositions. Inparticular embodiments, the disclosure provides compositions comprisingplatinum-based drug complexes and to methods of making and using thesecompositions. In some embodiments, the disclosure provides a compositioncomprising a complex of a therapeutic agent such as a platinum-baseddrug therapeutic agent and one or more polyglutamate molecules. In someembodiments, the disclosure provides a composition comprising a complexof a therapeutic agent such as a platinum-based drug therapeutic agentand cyclodextrin. In some embodiments, the compositions comprise acomplex containing a platinum-based drug complex. In some embodiments,the platinum-based drug complex comprises cisplatin or a salt thereof.In some embodiments, the platinum-based drug complex comprisesoxaliplatin, or a salt thereof. In some embodiments, the platinum-baseddrug complex comprises stratoplatin, paraplatin, platinol, cycloplatin,dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin,iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin,enloplatin, JM-216, 254-S, NK121, CI-973, DWA 2114R, NDDP, ordedaplatin, or a salt thereof. Compositions comprising delivery vehiclessuch as liposomes that contain/encapsulate the therapeutic agentcomplexes are also provided, as are methods of making and using theprovided compositions to treat hyperproliferative diseases such ascancer. In some embodiments, the disclosure provides liposomecompositions that comprise a liposome encapsulating a complex of aplatinum-based chemotherapeutic agent (e.g., cisplatin and oxaliplatin,or a salt thereof) and one or more polyglutamate molecule(s) (e.g., apolyglutamate having the structure (α-L-glutamyl)_(n).(γ-L-glutamyl)_(n), (α-D-glutamyl)_(n) and (γ-D-glutamyl)_(n), wheren=1, 2, 3, 4, 5, 2-8, 2-10, 4-6, or >5; and molecules such aspolyglutamated folates or polyglutamated antifolates containing thesepolyglutamate structures). In some embodiments, the liposome compositioncomprises a liposome encapsulating a complex of a platinum-basedchemotherapeutic agent (e.g., cisplatin, and oxaliplatin, or a saltthereof) and cyclodextrin (e.g., a derivatized beta cyclodextrin). Insome embodiments, the liposome is pegylated. In additional embodiments,the liposome composition further comprises one or more pharmaceuticallyacceptable carriers.

In one embodiment, the disclosure provides a composition comprising adelivery vehicle, such as a liposome composition that comprises apegylated liposome encapsulating a complex of a therapeutic agent or asalt thereof, and one or more polyglutamate molecule(s). In someembodiments, the therapeutic agent complex comprises a polyglutamatehaving the structure (α-L-glutamyl)_(n), where n=1, 2, 3, 4, 5, 2-8,2-10, 4-6, or >5. In some embodiments, the therapeutic agent complexcomprises a molecule containing the structure X-(α-L-glutamyl)_(n),where n=1, 2, 3, 4, 5, 2-8, 2-10, 4-6, or >5, and X is the structure ofthe non-therapeutic agent molecule(s) in the complex. In furtherembodiments, the therapeutic agent of the complex is a platinum-baseddrug agent. In one embodiment, the platinum-based chemotherapeutic agentis cisplatin. In another embodiment, the platinum-based chemotherapeuticagent is a cisplatin analog. In an additional embodiment, theplatinum-based chemotherapeutic agent is oxaliplatin. In an additionalembodiment, the platinum-based chemotherapeutic agent is selected fromthe group: nedaplatin, heptaplatin, and lobaplatin. In anotherembodiment, the platinum-based chemotherapeutic agent is selected fromthe group: nedaplatin, heptaplatin, and lobaplatin. In an additionalembodiment, the platinum-based chemotherapeutic agent is selected fromthe group: stratoplatin, paraplatin, platinol, cycloplatin,dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin,iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin,enloplatin, JM-216, 254-S, NK 121, CI-973, DWA 2114R, NDDP, anddedaplatin

In further embodiments, the disclosure provides a composition comprisinga delivery vehicle, such as a liposome composition that comprises apegylated liposome encapsulating a complex of a therapeutic agent or asalt thereof, and the therapeutic agent complex comprises apolyglutamated antifolate having the structureAntifolate-(α-L-glutamyl)_(n), where n=2-8, and a second therapeuticagent that is not a polyglutamated antifolate. In further embodiments,the second therapeutic agent is a platinum-based drug agent. In oneembodiment, the platinum-based chemotherapeutic agent is cisplatin. Inanother embodiment, the platinum-based chemotherapeutic agent is acisplatin analog. In an additional embodiment, the platinum-basedchemotherapeutic agent is oxaliplatin. In an additional embodiment, theplatinum-based chemotherapeutic agent is selected from the group:nedaplatin, heptaplatin, and lobaplatin. In another embodiment, theplatinum-based chemotherapeutic agent is selected from the group:nedaplatin, heptaplatin, and lobaplatin. In an additional embodiment,the platinum-based chemotherapeutic agent is selected from the group:stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin.

In another embodiment, the disclosure provides a composition comprisinga delivery vehicle, such as a liposome composition that comprises apegylated liposome encapsulating a complex of a therapeutic agent or asalt thereof, and the therapeutic agent complex comprises apolyglutamate having the structure (γ-L-glutamyl)_(n),(α-D-glutamyl)_(n), or (γ-D-glutamyl)_(n), where n=2-8. In someembodiments, the therapeutic agent-polyglutamate complex comprises amolecule containing the structure X-(γ-L-glutamyl)_(n),X-(α-D-glutamyl)_(n), or X_(γ-D-glutamyl)_(n), where n=1, 2, 3, 4, 5,2-8, 2-10, 4-6, or >5, and X is the structure of the non-therapeuticagent molecule(s) in the complex. In further embodiments, thetherapeutic agent of the complex is a platinum-based drug agent. In oneembodiment, the platinum-based chemotherapeutic agent is cisplatin. Inanother embodiment, the platinum-based chemotherapeutic agent is acisplatin analog. In an additional embodiment, the platinum-basedchemotherapeutic agent is oxaliplatin. In an additional embodiment, theplatinum-based chemotherapeutic agent is selected from the group:nedaplatin, heptaplatin, and lobaplatin. In another embodiment, theplatinum-based chemotherapeutic agent is selected from the group:nedaplatin, heptaplatin, and lobaplatin. In an additional embodiment,the platinum-based chemotherapeutic agent is selected from the group:stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin.

In further embodiments, the disclosure provides a composition comprisinga delivery vehicle, such as a liposome composition that comprises apegylated liposome encapsulating a complex that comprises apolyglutamated antifolate having the structureAntifolate-(γ-L-glutamyl)_(n), Antifolate-(α-D-glutamyl)_(n), orAntifolate-(γ-D-glutamyl)_(n), where n=1, 2, 3, 4, 5, 2-8, 2-10, 4-6,or >5, and a second therapeutic agent that is not a polyglutamatedantifolate. In further embodiments, the second therapeutic agent is aplatinum-based drug agent. In one embodiment, the platinum-basedchemotherapeutic agent is cisplatin. In another embodiment, theplatinum-based chemotherapeutic agent is a cisplatin analog. In anadditional embodiment, the platinum-based chemotherapeutic agent isoxaliplatin. In an additional embodiment, the platinum-basedchemotherapeutic agent is selected from the group: nedaplatin,heptaplatin, and lobaplatin. In another embodiment, the platinum-basedchemotherapeutic agent is selected from the group: nedaplatin,heptaplatin, and lobaplatin. In an additional embodiment, theplatinum-based chemotherapeutic agent is selected from the group:stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin.

In some embodiments, the therapeutic agent-polyglutamate complexcomprises a polyglutamated antifolate (e.g., having the structureAntifolate-[glutamyl]_(n), where n=1, 2, 3, 4, 5, 2-8, 2-10, 4-6, or >5;where [glutamyl]_(n) refers to the glutamyl attached to the antifolate,n is the number of attached glutamyls, and “Antifolate” is apolyglutamatable antifolate), and the therapeutic agent is not apolyglutamated antifolate. In some embodiments, the therapeutic agent isa platinum-based drug agent. In one embodiment, the platinum-basedchemotherapeutic agent is cisplatin. In another embodiment, theplatinum-based chemotherapeutic agent is a cisplatin analog. In anadditional embodiment, the platinum-based chemotherapeutic agent iscarboplatin. In an additional embodiment, the platinum-basedchemotherapeutic agent is oxaliplatin. In an additional embodiment, theplatinum-based chemotherapeutic agent is selected from the group:nedaplatin, heptaplatin, and lobaplatin. In another embodiment, theplatinum-based chemotherapeutic agent is selected from the group:nedaplatin, heptaplatin, and lobaplatin. In an additional embodiment,the platinum-based chemotherapeutic agent is selected from the group:stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin.

In some embodiments, the therapeutic agent-polyglutamate complexcomprises a polyglutamated folate (e.g., having the structureFolate-[glutamyl]_(n), where n=1, 2, 3, 4, 5, 2-8, 2-10, 4-6, or >5; andwherein -[glutamyl]_(n) refers to the glutamyl attached to the folateand n is the number of attached glutamates, and the therapeutic agent isnot polyglutamated folate. In some embodiments, the therapeutic agent isa platinum-based drug agent. In one embodiment, the platinum-basedchemotherapeutic agent is cisplatin. In another embodiment, theplatinum-based chemotherapeutic agent is a cisplatin analog. In anadditional embodiment, the platinum-based chemotherapeutic agent isoxaliplatin. In an additional embodiment, the platinum-basedchemotherapeutic agent is selected from the group: nedaplatin,heptaplatin, and lobaplatin. In another embodiment, the platinum-basedchemotherapeutic agent is selected from the group: nedaplatin,heptaplatin, and lobaplatin. In an additional embodiment, theplatinum-based chemotherapeutic agent is selected from the group:stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin.

In additional embodiments, the disclosure provides a compositioncomprising a delivery vehicle, such as a liposome composition thatcomprises a pegylated liposome encapsulating a complex of a therapeuticagent or a salt thereof, and a cyclodextrin.

In one embodiment, the disclosure provides a liposome compositioncomprising a pegylated liposome encapsulating a complex of aplatinum-based chemotherapeutic agent or a salt thereof and acyclodextrin. In some embodiments, the platinum-cyclodextrin complexcomprises cisplatin. In other embodiments, the platinum-cyclodextrincomplex comprises a cisplatin analog. In other embodiments, theplatinum-cyclodextrin complex comprises oxaliplatin. In additionalembodiments, the platinum-cyclodextrin complex comprises aplatinum-based chemotherapeutic agent selected from the group:nedaplatin, heptaplatin, and lobaplatin. In additional embodiments, theplatinum-cyclodextrin complex comprises a platinum-basedchemotherapeutic agent selected from the group: stratoplatin,paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin,picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin,platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK 121, CI-973,DWA 2114R, NDDP, and dedaplatin.

The cyclodextrin (CD) complexed with the platinum-based chemotherapeuticagent in the provided liposome compositions can be derivatized orunderivatized. In some embodiments, the cyclodextrin is derivatized. Infurther embodiments, the cyclodextrin is a derivatized beta-cyclodextrin(e.g., a hydroxypropyl beta-cyclodextrin (HP-beta-CD), and a sulfobutylether beta-CD (SBE)-beta-cyclodextrin). In some embodiments, thecyclodextrin is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more 2-hydroxylpropyl-3- group substitutions ofhydroxy groups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkylether group substitutions of hydroxy groups. In further embodiments, thecyclodextrin is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more sulfobutyl ether group substitutions ofhydroxy groups.

In some embodiments, the cyclodextrin complexed with the platinum-basedchemotherapeutic agent in the provided liposome compositions is aderivatized cyclodextrin of Formula I:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branched C₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group.

In some embodiments, the cyclodextrin complexed with the platinum-basedchemotherapeutic agent is a derivatized cyclodextrin of Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the pharmaceutically acceptable cationis selected from: an alkali metal such as Li⁺, Na⁺, or K⁺; an alkalineearth metal such as Ca⁺², or Mg⁺², and ammonium ions and amine cationssuch as the cations of (C1-C6)-alkylamines, piperidine, pyrazine,(C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine.

In some embodiments, liposomes in the provide liposome composition areanionic or neutral. In further embodiments, liposomes in the compositionhave a zeta potential that is less than or equal to zero. In furtherembodiments, liposomes in the composition has a zeta potential that isbetween 0 to −150 mV. In other embodiments, liposomes in the compositionare cationic. In further embodiments, liposomes in the composition havea zeta potential that is between 1 and 100 mV.

In some embodiments, liposomes in the provided liposome compositions arepegylated. In some embodiments, the polyethylene glycol of the pegylatedliposomes has a number average molecular weight of 200 to 5000 daltons.In some embodiments, the liposome compositions comprise pegylatedliposomes containing an internal phase having a pH in the range of2.8-6.8. In some embodiments, the internal phase of pegylated liposomesin the liposome composition comprise trehalose (e.g., 5% to 20% weightof trehalose).

In additional embodiments, the liposome composition comprises liposomesthat contain a targeting moiety. In some embodiments, the targetingmoiety is attached to one or both of a PEG and the exterior of theliposome. In additional embodiments, the targeting moiety has a specificaffinity for a surface antigen on a target cell of interest. In someembodiments, the targeting moiety is a polypeptide. In additionalembodiments, the targeting moiety is an antibody, a humanized antibody,an antigen binding fragment of an antibody, a single chain antibody, asingle-domain antibody, a bi-specific antibody, a synthetic antibody, apegylated antibody, or a multimeric antibody. In some embodiments, theliposome composition comprises liposomes that contain from 30 to 500targeting moieties. In further embodiments, the liposome compositioncomprises liposomes that contain from 30 to 200 targeting moieties.

In some embodiments, the liposome composition comprises liposomes thatcontain one or more of an immunostimulatory agent, a detectable markerand a maleimide disposed on at least one of the PEG and the exterior ofthe liposome.

In additional embodiments, the provided liposome compositions comprise apegylated liposome that further comprise a second complex formed by atherapeutic agent or a salt, acid or free base form thereof, and one ormore polyglutamate molecules. In some embodiments, the therapeutic agentof the second complex is a chemotherapeutic agent, an antimetabolite,and/or a taxane. In some embodiments, the therapeutic agent of thesecond complex is gemcitabine, a gemcitabine-based therapeutic agent,doxorubicin, a doxorubicin-based therapeutic agent, an antifolate, anantifolate-based chemotherapeutic, or a salt, acid or free base formthereof.

In some embodiments, the provided liposome compositions comprise apegylated liposome that further comprises a second complex formed by atherapeutic agent or a salt, acid or free base form thereof, and one ormore polyglutamate molecules. In some embodiments, the therapeutic agentof the second complex is a chemotherapeutic agent, an antimetabolite,and/or a taxane. In some embodiments, the therapeutic agent of thesecond complex is a member selected from the group: gemcitabine, agemcitabine-based therapeutic agent, doxorubicin, an antifolate, anantifolate-based chemotherapeutic, or a salt, acid or free base formthereof.

In additional embodiments, the provided liposome compositions comprisesa pegylated liposome that further comprises a second complex formed by atherapeutic agent or a salt, acid or free base form thereof, and acyclodextrin. In some embodiments, the therapeutic agent of the secondcomplex is a member selected from the group: gemcitabine, agemcitabine-based therapeutic agent, doxorubicin, an antifolate, anantifolate-based chemotherapeutic, or a salt, acid or free base formthereof. The cyclodextrin of the second complex can be derivatized orunderivatized. In some embodiments, the cyclodextrin is derivatized. Infurther embodiments, the cyclodextrin is a derivatized beta-cyclodextrin(e.g., a hydroxypropyl beta-cyclodextrin (HP-beta-CD), and a sulfobutylether beta-CD (SBE)-beta-cyclodextrin). In some embodiments, thecyclodextrin of the second complex is a derivatized beta-cyclodextrincomprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2-hydroxylpropyl-3-group substitutions of hydroxy groups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more sulfoalkyl ether group substitutions of hydroxy groups. Infurther embodiments, the cyclodextrin of the second complex is aderivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more sulfobutyl ether group substitutions of hydroxy groups.

In some embodiments, the cyclodextrin of the second complex contained inthe liposomes of the liposome composition (i.e., the complex formed by atherapeutic agent or a salt thereof, and a cyclodextrin) is aderivatized cyclodextrin of Formula I:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branched C₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group.

In some embodiments, the cyclodextrin of the second complex contained inthe liposomes of the liposome composition is a derivatized cyclodextrinof Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺², and ammonium ions andamine cations such as the cations of (C1-C6)-alkylamines, piperidine,pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine.

In some embodiments, liposomes in the liposome composition comprisebetween 100 to 100,000 of the second complex formed by the therapeuticagent or a salt thereof, and the cyclodextrin. In some embodiments, theliposome composition comprises liposomes that have a diameter in therange of 20 nm (nanometer) to 200 nm. In some embodiments, the liposomecomposition comprises liposomes that have a diameter in the range of forexample, 10-250 nm. In some embodiments, the liposomes have a diameterin the range of for example, 30-150 nm. In other embodiments, theliposomes have a diameter in the range of 40-70 nm. In some embodiments,liposomes in the composition comprise between 100 to 100,000platinum-based chemotherapeutic agent/cyclodextrin complexes. In someembodiments, the cyclodextrin of the second complex is different fromthe cyclodextrin of the platinum-based chemotherapeutic agent complex.In some embodiments, the liposome comprises the same cyclodextrin in thesecond complex and the platinum-based chemotherapeutic agent complex. Insome embodiments, the liposome comprises a cyclodextrin in the secondcomplex that is different from the cyclodextrin of the platinum-basedchemotherapeutic agent complex.

According to some embodiments, the provided liposome compositionsfurther comprise one or more of an immunostimulatory agent, a detectablemarker and a maleimide, wherein the immunostimulatory agent, thedetectable marker or the maleimide is attached to the PEG or theexterior of the liposome. In some embodiments, the immunostimulatingagent is at least one member selected from the group: a proteinimmunostimulating agent; a nucleic acid immunostimulating agent; achemical immunostimulating agent; a hapten; and an adjuvant. In someembodiments, the immunostimulating agent is at least one selected fromthe group: a fluorescein; a fluorescein isothiocyanate (FITC); a DNP; abeta glucan; a beta-1,3-glucan; and a beta-1,6-glucan. In someembodiments, the immunostimulatory agent and the detectable marker isthe same. In some embodiments, the liposome composition comprises ahapten. In further embodiments, the hapten comprises one or more offluorescein or Beta 1,6-glucan.

In some embodiments, the liposomes of the provided liposome compositionsfurther comprise at least one cryoprotectant selected from the groupconsisting of mannitol; trehalose; sorbitol; and sucrose. In additionalembodiments, the provided liposomal composition is in unit dosage form.In some embodiments, pharmaceutical compositions comprising the liposomecompositions disclosed herein are provided.

In some embodiments, the disclosure is directed to the use of theprovided liposome compositions in the treatment of disease. In someembodiments, the disclosure is directed to use of the provided liposomecompositions in the manufacture of a medicament for the treatment ofdisease.

In some embodiments, the disclosure provides a method of killing ahyperproliferative cell that comprises contacting a hyperproliferativecell with a liposome composition provided herein. In furtherembodiments, the hyperproliferative cell is a cancer cell.

In some embodiments, the disclosure provides a method for treating orpreventing disease in a subject needing such treatment or prevention,the method comprising administering an effective amount of a liposomecomposition provided herein to a subject in need thereof. In someembodiments, the disease is a hyperproliferative disorder such ascancer. In some embodiments, the administration is parenteral. In someembodiments, the administration is intravenous. In some embodiments, theadministration is subcutaneous.

In additional embodiments, the disclosure provides a method for treatingcancer in a subject, comprising administering an effective amount of aliposome composition disclosed herein to a subject having or at risk ofhaving cancer. In further embodiments, the cancer is a member selectedfrom the group: lung cancer, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma; and a hematologicmalignancy such as for example, a leukemia, a lymphoma and other B cellmalignancies, myeloma and other plasma cell dyscrasias. In someembodiments, the administration is parenteral. In some embodiments, theadministration is intravenous. In some embodiments, the administrationis subcutaneous.

In other embodiments, the disclosure provides a method for treatingcancer in a subject, comprising administering an effective amount of aliposome composition to a tumor expressing an antigen on its surface,the method comprising: administering a liposome composition disclosedherein to a subject having a tumor expressing the antigen in an amountto deliver an effective dose of the liposome composition to the tumor.In some embodiments, the administration is parenteral. In someembodiments, the administration is intravenous. In some embodiments, theadministration is subcutaneous.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomecomposition provided herein to a subject, wherein the liposome comprisesa targeting moiety with specific affinity for an antigen expressed onthe surface of a cancer cell, and wherein the subject has or is at riskof having a cancer cell that expresses the antigen. In furtherembodiments, the antigen is a folate receptor. In some embodiments, theadministration is parenteral. In some embodiments, the administration isintravenous. In some embodiments, the administration is subcutaneous

Also provided is maintenance therapy that comprise administering aneffective amount of a liposome composition disclosed herein to a subjectthat is undergoing or has undergone cancer therapy. In some embodiments,the administration is parenteral. In some embodiments, theadministration is intravenous. In some embodiments, the administrationis subcutaneous.

In another aspect, a kit comprising a liposome composition providedherein, and instructions for use, is provided. In some embodiments, thekit comprises a container comprising a liposome composition disclosedherein. In some embodiments, the kit further comprises a secondcontainer comprising a second liposome composition disclosed herein or apharmaceutically acceptable carrier.

In some embodiments, the disclosure provides:

-   [1] a liposome composition comprising a liposome encapsulating    -   (a) a complex of a platinum-based chemotherapeutic agent or a        salt thereof and one or more polyglutamates or    -   (b) a complex of a platinum-based chemotherapeutic agent or a        salt thereof and a cyclodextrin;    -   wherein the liposome comprises one or more pharmaceutically        acceptable carriers; and a pegylated liposome;-   [2] the liposome composition of [1], wherein the platinum-based    chemotherapeutic agent is cisplatin or a cisplatin analog;-   [3] the liposome composition according to [1] or [2], wherein the    platinum-based chemotherapeutic agent is a member selected from the    group: cisplatin, oxaliplatin, stratoplatin, paraplatin, platinol,    cycloplatin, dexormaplatin, spiroplatin picoplatin, nedaplatin,    triplatin, tetraplatin, lipoplatin, lobaplatin, ormaplatin,    zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S,    NK 121, CI-973, DWA2114R, NDDP, and dedaplatin;-   [4] the liposome composition according to any of [1]-[3], wherein    the liposome encapsulates a complex of a platinum-based    chemotherapeutic agent or a salt thereof and a cyclodextrin, and    wherein the cyclodextrin is a derivatized or underivatized    beta-cyclodextrin;-   [5] the liposome composition of [4], wherein the cyclodextrin is a    derivatized beta-cyclodextrin;-   [6] the liposome composition of [4], wherein the cyclodextrin (CD)    is a hydroxypropyl beta-CD (HP-beta-CD), or a sulfobutyl ether    beta-CD ((SBE)-beta-CD);-   [7] the liposome composition of [5], wherein the cyclodextrin is a    derivatized beta-cyclodextrin comprising:    -   (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2-hydroxylpropyl-3-        group substitutions of hydroxy groups; or    -   (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkyl ether        group substitutions of hydroxy groups;-   [8] the liposome composition of [7], wherein the cyclodextrin is a    derivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9,    10, or more sulfobutyl ether group substitutions of hydroxy groups;-   [9] the liposome composition of [5], wherein the cyclodextrin is a    derivatized beta-cyclodextrin of Formula III:

-   -   wherein R equals:    -   (a) (H)_(21-X) or (—(CH₂)₄-SO₃Na)_(x), and x=1.0-10.0, 1.0-5.0,        6.0-7.0 or 8.0-10.0;    -   (b) (H)_(21-X) or (—(CH₂CH(OH)CH₃)_(x), and x=1.0-10.0, 1.0-5.0,        6.0-7.0 or 8.0-10.0;    -   (c) (H)_(21-X) or (sulfoalkyl ether)_(x), and x=1.0-10.0,        1.0-5.0, 6.0-7.0 or 8.0-10.0; or (H)_(21-X) or        (—(CH₂)₄—SO₃Na)_(x), and x=1.0-10.0, 1.0-5.0, 6.0-7.0 or        8.0-10.0;

-   [10] the liposome composition according to any of [1]-[9], wherein    the cyclodextrin is a derivatized cyclodextrin of Formula I:

-   -   wherein: n is 4, 5, or 6; and    -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each,        independently, —H, a straight chain or branched C₁-C₈- alkylene        group, a 2-hydroxylpropyl-3- group; or an optionally substituted        straight-chain or branched C₁-C₆ group, wherein at least one of        R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or        branched C₁-C₈- alkylene group or a 2-hydroxylpropyl-3- group;

-   [11] the liposome composition of according to any of [1]-[9],    wherein the cyclodextrin is a derivatized cyclodextrin of Formula    II:

-   -   wherein: n is 4, 5, or 6; and    -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each,        independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻ group; wherein        at least one of R₁ and R₂ is independently a —O—(C₂-C₆        alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and        S₉ are each, independently, a —H or a H or a pharmaceutically        acceptable cation;

-   [12] the liposome composition of [11], wherein the pharmaceutically    acceptable cation is selected from: an alkali metal such as Li⁺,    Na⁺, or K⁺; an alkaline earth metal such as Ca⁺², or Mg⁺²; and    ammonium ions and amine cations such as the cations of    (C₁-C₆)-alkylamines, piperidine, pyrazine, (C₁-C₆)-alkanolamine, and    (C₄-C₈)-cycloalkanolamin;

-   [13] the liposome composition according to any of [1]-[12], wherein    each liposome comprises between 100 to 100,000 complexes formed by    the platinum-based chemotherapeutic agent or salt thereof, and the    cyclodextrin;

-   [14] the liposome composition according to any of [1]-[13], wherein    the liposome has a diameter in the range of 20 nm to 200 nm;

-   [15] the liposome composition according to any of [1]-[13], wherein    the liposome has a diameter in the range of 80 nm to 120 nm;

-   [16] the liposome composition according to any of [1]-[15], wherein    the polyethylene glycol of the liposome has a number average    molecular weight (Mn) of 200 to 5000 daltons;

-   [17] the liposome composition according to any of [1]-[16], wherein    the liposome comprises a steric stabilizer;

-   [18] the liposome composition of [17], which comprises a steric    stabilizer selected from the group consisting of polyethylene glycol    (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl    pyrrolidone) (PVP); poly(acrylamide) (PAA);    poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl    polyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic    poly-N-vinylpyrrolidones; L-amino-acid-based polymer; and polyvinyl    alcohol;

-   [19] the liposome composition according to any of [1]-[18], wherein    the liposome comprises at least one of an anionic lipid and a    neutral lipid;

-   [20] the liposome composition according to any of [1]-[19], wherein    the liposome comprises at least one is a member selected from the    group: DSPE; DSPE-PEG-maleimide; DSPE-PEG-FITC; HSPC; HSPC-PEG;    cholesterol; cholesterol-PEG; and cholesterol-maleimide;

-   [21] the liposome composition according to any of [1]-[20], wherein    the liposome comprises oxidized phospholipids, optionally wherein    the phospholipids are a member selected from the group consisting of    phosphatidylserines, phosphatidylinositols,    phosphatidylethanolamines, phosphatidylcholines and    1-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate;

-   [22] the liposome composition according to any of [1]-[20], wherein    the liposome comprises oxidized    1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylchloine (OxPAPC),    optionally wherein the oxPAPCs are epoxyisoprostane-containing    phospholipids;

-   [23] the liposome composition of [22], wherein the oxPAPC is    1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine    (5,6-PEIPC),    1-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine    (PECPC) and/or 1-palmitoyl-2-(epoxy-isoprostane    E2)-sn-glycero-4-phosphocholine (PEIPC);

-   [24] the liposome composition according to any of [1]-[23], wherein    the liposome is anionic or neutral;

-   [25] the liposome composition according to any of [1]-[24], wherein    the liposome has a zeta potential that is less than or equal to    zero;

-   [26] the liposome composition of [25], wherein the liposome has a    zeta potential that is between 0 to −150 mV or between −30 to −50    mV;

-   [27] the liposome composition according to any of [1]-[23], wherein    the liposome has a zeta potential that is greater than zero;

-   [28] the liposome composition of [27], wherein the liposome has a    zeta potential that is between 1 and 100 mV, between 5 to 60 mV, or    between 10 to 50 mV;

-   [29] the liposome composition according to any of [1]-[23], [27], or    [28], wherein the liposome is cationic;

-   [30] the liposome composition according to any of [1]-[29], wherein    the pH of the internal phase of the liposome is between [25] and    [75]

-   [31] the liposome composition of [30], wherein the pharmaceutically    acceptable carrier comprises citrate buffer at a concentration of    between 5 to 200 mM and a pH of between 2]8 to 6 or a total    concentration of sodium acetate and calcium acetate of between 50 mM    to 500 mM;

-   [32] the liposome composition according to any of [1]-[31], wherein    the liposome internal phase comprises trehalose;

-   [33] the liposome composition of [32], wherein the liposome    comprises 5% to 20% weight of trehalose;

-   [34] the liposome composition according to any of [1]-[33], which    further comprises a targeting moiety attached to one or both of a    PEG and the exterior of the liposome, and wherein the targeting    moiety has a specific affinity for a surface antigen on a target    cell of interest;

-   [35] the liposome composition of [34], wherein the targeting moiety    is attached to one or both of the PEG and the exterior of the    liposome by a covalent bond;

-   [36] the liposome composition according to [34] or [35], wherein the    targeting moiety is a polypeptide;

-   [37] the liposome composition according to any of [34]-[36], wherein    the targeting moiety is an antibody or a fragment of an antibody;

-   [38] the liposome composition according to any of [34]-[37], wherein    the targeting moiety binds the surface antigen with an equilibrium    dissociation constant (Kd) in a range of 0.5×10⁻¹⁰ to 10×10⁻⁶ as    determined using BIACORE® analysis;

-   [39] the liposome composition according to any of [34]-[38], wherein    the targeting moiety specifically binds one or more folate receptors    selected from the group: folate receptor alpha (FR-α), folate    receptor beta (FR-β), and folate receptor delta (FR-δ);

-   [40] the liposome composition according to any of [34]-[39], wherein    the targeting moiety comprises one or more members selected from the    group: an antibody, a humanized antibody, an antigen binding    fragment of an antibody, a single chain antibody, a single-domain    antibody, a bi-specific antibody, a synthetic antibody, a pegylated    antibody, and a multimeric antibody;

-   [41] the liposome composition according to any of [34]-[40], wherein    each PEGylated liposome comprises from 30 to 500 targeting moieties;

-   [42] the liposome composition according to any of [1]-[33], wherein    the liposome does not comprise a targeting moiety attached to one or    both of a PEG and the exterior of the liposome;

-   [43] the liposome composition of [41], wherein each PEGylated    liposome comprises from 30 to 200 targeting moieties;

-   [44] the liposome composition according to any of [1]-[43], wherein    the liposome further comprises a second complex formed by a    therapeutic agent or a salt thereof, and one or more polylglutamate    molecules or a cyclodextrin;

-   [45] the liposome composition of [44], wherein the therapeutic agent    of the second complex is gemcitabine or doxorubicin, or a salt    thereof;

-   [46] the liposome composition according to [44] or [45], wherein the    second complex comprises cyclodextrin and wherein the cyclodextrin    of the second complex is a derivatized or underivatized    cyclodextrin;

-   [47] the liposome composition of [46], wherein the cyclodextrin of    the second complex is a derivatized beta-cyclodextrin;

-   [48] the liposome composition of [47], wherein the derivatized    beta-cyclodextrin is a hydroxypropyl beta-cyclodextrin (HP-beta-CD),    or a sulfobutyl ether beta-cyclodextrin (SBE)-beta-CD;

-   [49] the liposome composition according to any of [46]-[48], wherein    the cyclodextrin of the second complex is a derivatized    beta-cyclodextrin comprising:    -   (a) 1, 2, 3, 4, 5, 6, or more 2-hydroxylpropyl-3- group        substitutions of hydroxy groups; or    -   (b) 1, 2, 3, 4, 5, 6, or more sulfoalkyl ether group        substitutions of hydroxy groups;

-   [50] the liposome composition of [49], wherein the cyclodextrin of    the second complex is a derivatized beta-cyclodextrin comprising 1,    2, 3, 4, 5, 6, or more sulfobutyl ether group substitutions of    hydroxy groups;

-   [51] the liposome composition according to any of [44]-[50], wherein    the cyclodextrin of the second complex is a derivatized    beta-cyclodextrin of Formula III:

-   -   wherein R equals:    -   (a) (H)_(21-X) or (—(CH₂)₄—SO₃Na)_(x), and x=1.0-10.0, 1.0-5.0,        6.0-7.0 or 8.0-10.0;    -   (b) (H)_(21-X) or (—(CH₂CH(OH)CH₃)_(x), and x=1.0-10.0, 1.0-5.0,        6.0-7.0 or 8.0-10.0;    -   (c) (H)_(21-X) or (sulfoalkyl ether)_(x), and x=1.0-10.0,        1.0-5.0, 6.0-7.0 or 8.0-10.0; or    -   (d) (H)_(21-X) or (—(CH₂)₄—SO₃Na)_(x), and x=1.0-10.0, 1.0-5.0,        6.0-7.0 or 8.0-10.0;

-   [52] the liposome composition according to any of [44]-[51], wherein    the cyclodextrin of the second complex is a derivatized cyclodextrin    of Formula I:

-   -   wherein: n is 4, 5, or 6;    -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each,        independently, —H, a straight chain or branched C₁-C₈- alkylene        group, a 2-hydroxylpropyl-3- group; or an optionally substituted        straight-chain or branched C₁-C₆ group, wherein at least one of        R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or        branched C₁-C₈- alkylene group or a 2-hydroxylpropyl-3- group;

-   [53] the liposome composition according to any of [44]-[51], wherein    the cyclodextrin of the second complex is a derivatized cyclodextrin    of formula II:

-   -   wherein: n is 4, 5, or 6;    -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each,        independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻ group; wherein        at least one of R₁ and R₂ is independently a —O—(C₂-C₆        alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and        S₉ are each, independently, a —H or a pharmaceutically        acceptable cation;

-   [54] the liposome composition of [53], wherein the pharmaceutically    acceptable cation is selected from: an alkali metal such as Li⁺,    Na⁺, or K⁺; an alkaline earth metal such as Ca⁺², or Mg+²; and    ammonium ions and amine cations such as the cations of    (C₁-C₆)-alkylamines, piperidine, pyrazine, (C₁-C₆)-alkanolamine, and    (C₄-C₈)-cycloalkanolamine;

-   [55] the liposome composition according to any of [46]-[54], wherein    each liposome comprises between 100 to 100,000 of the second complex    formed by the therapeutic agent or a salt thereof, and the    cyclodextrin;

-   [56] the liposome composition according to any of [46]-[55], wherein    the cyclodextrin of the second complex is different from the    cyclodextrin of the platinum-based chemotherapeutic agent complex;

-   [57] the liposome composition according to any of [46]-[55], wherein    the cyclodextrin of the second complex is the same as the    cyclodextrin of the platinum-based chemotherapeutic agent complex;

-   [58] the liposome composition according to any of [1]-[57], further    comprising one or more of an immunostimulatory agent, a detectable    marker and a maleimide, wherein the immunostimulatory agent, the    detectable marker or the maleimide is attached to the PEG or the    exterior of the liposome;

-   [59] the liposome composition of [58], wherein immunostimulating    agent is at least one is a member selected from the group: a protein    immunostimulating agent; a nucleic acid immunostimulating agent; a    chemical immunostimulating agent; a hapten; and an adjuvant;

-   [60] the liposome composition according to [58] or [59], wherein the    immunostimulating agent is at least one selected from the group: a    fluorescein; a fluorescein isothiocyanate (FITC); a DNP; a beta    glucan; a beta-1,3-glucan; and a beta-1,6-glucan;

-   [61] the liposome composition according to any of [58]-[60], wherein    the immunostimulatory agent and the detectable marker is the same;

-   [62] the liposome composition of [59], which comprises a hapten;

-   [63] the liposome composition of [62], wherein the hapten comprises    one or more of fluorescein or Beta 1,6-glucan;

-   [64] the liposome composition according to any of [1]-[63], which    further comprises at least one cryoprotectant selected from the    group consisting of mannitol; trehalose; sorbitol; and sucrose;

-   [65] the composition according to any of [1]-[64], which is in unit    dosage form;

-   [66] the liposome composition according to any of [1]-[65] for use    in the treatment of disease;

-   [67] Use of the liposome composition according to any of [1]-[65],    in the manufacture of a medicament for the treatment of disease;

-   [68] a method of killing a hyperproliferative cell comprising    contacting a hyperproliferative cell with the liposome composition    according to any of [1]-[65]

-   [69] the method of [68], wherein the hyperproliferative cell is a    cancer cell;

-   [70] a method for treating or preventing disease in a subject    needing such treatment or prevention, the method comprising    administering an effective amount of the liposome composition    according to any of [1]-[65] to a subject in need thereof;

-   [71] a method for treating cancer in a subject, comprising    administering an effective amount of a liposome composition    according to any of [1]-[65] to a subject having or at risk of    having cancer;

-   [72] the method of [71], wherein the cancer is a member selected    from the group: lung cancer, pancreatic cancer, breast cancer,    ovarian cancer, prostate cancer, head and neck cancer, gastric    cancer, gastrointestinal cancer, colorectal cancer, esophageal    cancer, cervical cancer, liver cancer, kidney cancer, biliary duct    cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,    osteosarcoma), brain cancer, central nervous system cancer, and    melanoma; and a hematologic malignancy such as for example, a    leukemia, a lymphoma and other B cell malignancies, myeloma and    other plasma cell dyscrasias;

-   [73] a method of delivering a liposome composition to a tumor    expressing an antigen on its surface, the method comprising:    administering the liposome composition according to any of [34]-[41]    or [43]-[65] to a subject having a tumor expressing the antigen    bound by the liposome targeting moiety in an amount to deliver a    therapeutically effective dose of the liposome composition to the    tumor;

-   [74] a method for treating cancer that comprises administering an    effective amount of the liposome composition according to any of    [34]-[41] or [43]-[65] to a subject having or at risk of having a    cancer cell that expresses on its surface the antigen bound by the    liposome targeting moiety;

-   [75] a maintenance therapy that comprises administering an effective    amount of the liposome composition of any of [1]-[65] to a subject    that is undergoing or has undergone cancer therapy;

-   [76] the method according to any of [70]-[75], wherein the    administration is parenteral;

-   [77] the method of [76], wherein the administration is intravenous;    and/or

-   [78] a pharmaceutical composition comprising the liposome    composition according to any of [1]-[65].

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides exemplary L-gamma polyglutamated antifolates.

FIG. 2 provides exemplary alpha and D-gamma polyglutamated antifolates.

FIGS. 3A and 3B depict the formation and dissolution of cisplatincomplexes with poly gamma glutamic acid (FIG. 3A) and poly alphaL-glutamic acid (FIG. 3B).

DETAILED DESCRIPTION

The disclosure generally relates to compositions comprising therapeuticagent complexes and to methods of making and using the compositions. Inparticular embodiments, the disclosure provides compositions comprisingplatinum-based drug complexes and to methods of making and using thesecompositions. In some embodiments, the disclosure provides a compositioncomprising a complex of a therapeutic agent such as a platinum-baseddrug therapeutic agent and one or more polyglutamate molecules. In someembodiments, the disclosure provides a composition comprising a complexof a therapeutic agent such as a platinum-based drug therapeutic agentand cyclodextrin. Methods of making and using the disclosed compositionsto treat diseases such as cancer are also provided, as are kitscontaining the compositions.

(a) Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

It is understood that wherever embodiments, are provided herein with thelanguage “comprising” otherwise analogous embodiments, described interms of “containing” “consisting of” and/or “consisting essentially of”are also provided. However, when used in the claims as transitionalphrases, each should be interpreted separately and in the appropriatelegal and factual context (e.g., in claims, the transitional phrase“comprising” is considered more of an open-ended phrase while“consisting of” is more exclusive and “consisting essentially of”achieves a middle ground).

As used herein, the singular form “a”, “an”, and “the”, includes pluralreferences unless it is expressly stated or is unambiguously clear fromthe context that such is not intended.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

The term “non-natural amino acid” refers to an amino acid that is not aproteinogenic amino acid, or a post-translationally modified variantthereof. In particular, the term refers to an amino acid that is not oneof the 20 common amino acids or pyrrolysine or selenocysteine, orpost-translationally modified variants thereof.

The terms “polyglutamate”, polyglutamated”, or variations thereof, asused herein, refer to a molecule comprising at least one chain of 2 ormore linked glutamyl groups. Polyglutamate chains can be linear orbranched. In some embodiments, the polyglutamate comprises 2, 3, 4, 5,6, 2-10, 4-6, or more than 5 glutamyl groups. Linear polyglutamatechains can contain for example, glutamyl groups containing either analpha carboxyl group or a gamma carboxyl group linkage. The term gammalinkage as it relates to a polyglutamate, refers to a peptide bondbetween the amino group of a first glutamyl residue and the carboxylgroup at the gamma carbon of the side chain of a second glutamyl residuein the polyglutamate. The term alpha linkage as it relates to apolyglutamate, refers to a peptide bond between the amino group of afirst glutamyl residue and the carboxyl group at the alpha carbon of theside chain of a second glutamyl residue in the polyglutamate. Theglutamyl groups in a polyglutamate can contain alpha linkages, gammalinkages, or a combination of alpha and gamma linkages. In someembodiments, the glutamyl groups in the polyglutamate contain only alphalinkages. In some embodiments, the glutamyl groups in the polyglutamatecontain only gamma linkages. In embodiments, each of the glutamyl groupsof a polyglutamate is an L isomer. The polyglutamates can contain Lglutamyl isomers, D, glutamyl isomers, or a combination thereof. Inparticular embodiments, each of the glutamyl groups of the polyglutamateis an L isomer. In some embodiments, each of the glutamyl groups of thepolyglutamate is an L isomer.

The term “polyglutamated antifolate” and iterations thereof, refers toan antifolate molecule that comprises at least one glutamyl group inaddition to the glutamyl group of the antifolate (i.e.,Antifolate-[glutamyl]n, where -[glutamyl]n refers to the glutamatesattached to the antifolate, n is the number of attached glutamates, andwherein n is greater than 1). Reference to the number of glutamyl groupsin an antifolate herein takes into account the glutamyl group ofantifolate or folate. For example, an Antifolate-[glutamyl]n compositionwhere n=5 is referred to herein as hexaglutamated Antifolate orAntifolate hexaglutamate. See, e.g., FIG. 1 and FIG. 2.

The term “polyglutamated folate” and iterations thereof, refers to afolate molecule that comprises at least one glutamyl group in additionto the glutamyl group of the folate (i.e., Folate-(glutamyl)n, where-[glutamyl]n refers to the glutamates attached to the folate, n is thenumber of attached glutamates, and wherein n is greater than 1).

Unless otherwise explicitly indicated herein, the provided therapeuticagent complexes (e.g., platinum-polyglutamate complexes andplatinum-cyclodextrin complexes) are non-covalent complexes wherein thebonds between the components of the complex are non-covalent bonds,i.e., weak bonds such as H-bonds and Van der Waals forces. The term“complex” as used herein includes, but is not limited to, an inclusioncomplex. The term “inclusion complex” as used herein refers to inclusioncomplexes wherein the platinum agent is surrounded by and entrappedwithin a cyclodextrin, and to partial inclusion complexes wherein theplatinum agent is surrounded partially by cyclodextrin.

As use herein, the term “isolated” refers to a composition which is in aform not found in nature. For example, isolated polyglutamate (e.g.,gamma-L-polyglutamate (L-γPGA) and alpha-L-polyglutamate (L-αPGA)compositions) include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, the polyglutamate (e.g., alpha L-γPGA or L-αPGA) which isisolated is substantially pure. Isolated compositions will be free orsubstantially free of material with which they are naturally associatedsuch as other cellular components such as proteins and nucleic acidswith which they may potentially be found in nature, or the environmentin which they are prepared (e.g., cell culture). The alphapolyglutamated compositions may be formulated with diluents or adjuvantsand still for practical purposes be isolated—for example, polyglutamatecompositions will normally be mixed with pharmaceutically acceptablecarriers or diluents when used in diagnosis or therapy. In someembodiments, the polyglutamate compositions (e.g., alpha L-γPGA orL-αPGA) and delivery vehicles such as liposomes containing thepolyglutamates contain less than 1% or less than 0.1% undesired DNA orprotein content. In some embodiments, the polyglutamate compositions(e.g., alpha L-γPGA or L-αPGA) are “isolated.”

The term “targeting moiety” is used herein to refer to a molecule thatprovides an enhanced affinity for a selected target, e.g., a cell, celltype, tissue, organ, region of the body, or a compartment, e.g., acellular, tissue or organ compartment. The targeting moiety can comprisea wide variety of entities. Targeting moieties can include naturallyoccurring molecules, or recombinant or synthetic molecules. In someembodiments, the targeting moiety is an antibody, antigen-bindingantibody fragment, bispecific antibody or other antibody-based moleculeor compound. In some embodiments, the targeting moiety is an aptamer,avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidinbinding pair, a peptide, protein, carbohydrate, lipid, vitamin, toxin, acomponent of a microorganism, a hormone, a receptor ligand or anyderivative thereof. Other targeting moieties are known in the art andare encompassed by the disclosure.

The terms “specific affinity” or “specifically binds” mean that atargeting moiety such as an antibody or antigen binding antibodyfragment, reacts or associates more frequently, more rapidly, withgreater duration, with greater affinity, or with some combination of theabove to the epitope, protein, or target molecule than with alternativesubstances, including proteins unrelated to the target epitope. Becauseof the sequence identity between homologous proteins in differentspecies, specific affinity can, in several embodiments, include abinding agent that recognizes a protein or target in more than onespecies. Likewise, because of homology within certain regions ofpolypeptide sequences of different proteins, the term “specificaffinity” or “specifically binds” can include a binding agent thatrecognizes more than one protein or target. It is understood that, incertain embodiments, a targeting moiety that specifically binds a firsttarget may or may not specifically bind a second target. As such,“specific affinity” does not necessarily require (although it caninclude) exclusive binding, e.g., binding to a single target. Thus, atargeting moiety may, in certain embodiments, specifically bind morethan one target. In certain embodiments, multiple targets may be boundby the same targeting moiety.

The terms “epitope” and “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody or bindingmoiety. When the antigen is a polypeptide, epitopes can be formed bothfrom contiguous amino acids and noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained upon protein denaturing, whereas epitopesformed by tertiary folding are typically lost upon protein denaturing.An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation.

Expressions like “binding affinity for a target”, “binding to a target”and analogous expressions known in the art refer to a property of atargeting moiety which may be directly measured through thedetermination of the affinity constants, e.g., the amount of targetingmoiety that associates and dissociates at a given antigen concentration.Different methods can be used to characterize the molecular interaction,such as, but not limited to, competition analysis, equilibrium analysisand microcalorimetric analysis, and real-time interaction analysis basedon surface plasmon resonance interaction (for example using a BIACORE®instrument). These methods are well-known to the skilled person and aredescribed, for example, in Neri et al., Tibtech 14:465-470 (1996), andJansson et al., J. Biol. Chem. 272:8189-8197 (1997).

The term “delivery vehicle” refers generally to any compositions thatacts to assist, promote or facilitate entry of the provided therapeuticagent complexes (e.g., platinum-polyglutamate complexes andplatinum-cyclodextrin complexes) into a cell. Such delivery vehicles areknown in the art and include, but are not limited to, liposomes,lipospheres, polymers (e.g., polymer-conjugates), peptides, proteinssuch as antibodies (e.g., immunoconjugates, such as Antibody DrugConjugates (ADCs)) and antigen binding antibody fragments andderivatives thereof), cellular components, cyclic oligosaccharides(e.g., cyclodextrins), micelles, microparticles (e.g., microspheres),nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles,and core-shell nanoparticles), hydrogels, lipoprotein particles, viralsequences, viral material, or lipid or liposome formulations, andcombinations thereof. The delivery vehicle can be linked directly orindirectly to a targeting moiety. In some examples, the targeting moietyis selected from among a macromolecule, a protein, a peptide, amonoclonal antibody or a fatty acid lipid.

A “subject” refers to a human or vertebrate mammal including but notlimited to a dog, cat, horse, goat and primate, e.g., monkey. Thus, theinvention can also be used to treat diseases or conditions in non-humansubjects. For instance, cancer is one of the leading causes of death incompanion animals (i.e., cats and dogs). In some embodiments, of theinvention, the subject is a human. In this disclosure, the term“subject” and “patient” is used interchangeably and has the samemeaning. It is preferred generally that a maximum dose be used, that is,the highest safe dose according to sound medical judgment.

As used herein an “effective amount” refers to a dosage of an agentsufficient to provide a medically desirable result. The effective amountwill vary with the desired outcome, the particular condition beingtreated or prevented, the age and physical condition of the subjectbeing treated, the severity of the condition, the duration of thetreatment, the nature of the concurrent or combination therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose. In the case of cancer, the effectiveamount of an agent may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the disorder. To the extent the drug mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy in vivo can be measuredfor example, by assessing the duration of survival, duration ofprogression free survival (PFS), the response rates (RR), duration ofresponse, and/or quality of life.

The terms “hyperproliferative disorder”, “hyperproliferative disease”,“proliferative disorder”, and “proliferative disease”, are usedinterchangeably herein to pertain to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo. In some embodiments, the proliferative disease is cancer ortumor disease (including benign or cancerous) and/or any metastases,wherever the cancer, tumor and/or the metastasis is located. In someembodiments, the proliferative disease is a benign or malignant tumor.In some embodiments, the proliferative disease is a non-cancerousdisease. In some embodiments, the proliferative disease is ahyperproliferative condition such as hyperplasias, fibrosis (especiallypulmonary, but also other types of fibrosis, such as renal fibrosis),angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferationin the blood vessels, such as stenosis or restenosis followingangioplasty.

“Cancer,” “tumor,” or “malignancy” are used as synonymous terms andrefer to any of a number of diseases that are characterized byuncontrolled, abnormal proliferation of cells, the ability of affectedcells to spread locally or through the bloodstream and lymphatic systemto other parts of the body (metastasize) as well as any of a number ofcharacteristic structural and/or molecular features. “Tumor,” as usedherein refers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues. A “cancerous tumor,” or “malignant cell” is understood as acell having specific structural properties, lacking differentiation andbeing capable of invasion and metastasis. A cancer that can be treatedusing a platinum-based chemotherapeutic agent provided herein includeswithout limitation, a non-hematologic malignancy including such as forexample, lung cancer, pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colorectal cancer, esophageal cancer, cervical cancer, livercancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladdercancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervoussystem cancer, and melanoma; and a hematologic malignancy such as forexample, a leukemia, a lymphoma and other B cell malignancies, myelomaand other plasma cell dyscrasias. Other types of cancer and tumors thatmay be treated using a platinum-based chemotherapeutic agent providedherein or otherwise known in the art. The terms “cancer,” “cancerous,”“cell proliferative disorder,” “proliferative disorder,” and “tumor” arenot mutually exclusive as referred to herein.

Terms such as “treating,” or “treatment,” or “to treat” refer to both(a) therapeutic measures that cure, slow down, lessen symptoms of,and/or halt progression of a diagnosed pathologic condition or disorderand (b) prophylactic or preventative measures that prevent and/or slowthe development of a targeted disease or condition. Thus, subjects inneed of treatment include those already with the cancer, disorder ordisease; those at risk of having the cancer or condition; and those inwhom the infection or condition is to be prevented. Subjects areidentified as “having or at risk of having” cancer, an infectiousdisease, a disorder of the immune system, a hyperproliferative disease,or another disease or disorder referred to herein using well-knownmedical and diagnostic techniques. In certain embodiments, a subject issuccessfully “treated” according to the methods provided herein if thesubject shows, e.g., total, partial, or transient amelioration orelimination of a symptom associated with the disease or condition (e.g.,cancer). In specific embodiments, the terms treating,” or “treatment,”or “to treat” refer to the amelioration of at least one measurablephysical parameter of a proliferative disorder, such as growth of atumor, not necessarily discernible by the patient. In other embodiments,the terms treating,” or “treatment,” or “to treat” refer to theinhibition of the progression of a proliferative disorder, eitherphysically by, e.g., stabilization of a discernible symptom,physiologically by, e.g., stabilization of a physical parameter, orboth. In other embodiments, the terms treating,” or “treatment,” or “totreat” refer to the reduction or stabilization of tumor size, tumor cellproliferation or survival, or cancerous cell count. Treatment can bewith a platinum-based chemotherapeutic agent, alone or in combinationwith an additional therapeutic agent. As used herein the terms “treat”,“treating” and “treatment” include administering the composition priorto the onset of clinical symptoms of a disease state/condition so as toprevent the development of any symptom, as well as administering thecomposition after the onset of one or more clinical symptoms of adisease state/condition so as to reduce or eliminate any such symptom,aspect or characteristic of the disease state/condition. Such treatingneed not be absolute to be useful.

“Subject” and “patient,” and “animal” are used interchangeably and referto mammals such as human patients and non-human primates, as well asexperimental animals such as rabbits, rats, and mice, and other animals.Animals include all vertebrates, e.g., mammals and non-mammals, such aschickens, amphibians, and reptiles. “Mammal” as used herein refers toany member of the class Mammalia, including, without limitation, humansand nonhuman primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs, and other members of theclass Mammalia known in the art. In a particular embodiment, the patientis a human.

The terms “treatment of a proliferative disorder”, “treatment of ahyperproliferative disorder” and iterations thereof, is used herein toinclude maintaining or decreasing tumor size, inducing tumor regression(either partial or complete), inhibiting tumor growth, and/or increasingthe life span of a subject having the proliferative disorder. In oneembodiment, the proliferative disorder is a solid tumor. Such tumorsinclude, for example, lung cancer, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma; and a hematologicmalignancy such as for example, a leukemia, a lymphoma and other B cellmalignancies, myeloma and other plasma cell dyscrasias.

The term “therapeutic agent” is used herein to refer to an agent or aderivative thereof that can interact with a hyperproliferative cell suchas a cancer cell or an immune cell, thereby reducing the proliferativestatus of the cell and/or killing the cell. Examples of therapeuticagents include, but are not limited to, chemotherapeutic agents,cytotoxic agents, platinum-based chemotherapeutic agents (e.g.,cisplatin and oxaliplatin), taxanes (e.g., TAXOL®), etoposide,alkylating agents (e.g., cyclophosphamide, ifosamide), metabolicantagonists (e.g., methotrexate (MTX), 5-fluorouracil gemcitabine, or aderivative thereof), antitumor antibiotics (e.g., mitomycin,doxorubicin), plant-derived antitumor agents (e.g., vincristine,vindesine, TAXOL®). Such agents may further include, but are not limitedto, the anticancer agents trimetrexate, temozolomide, raltitrexed,S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), 6-benzyguanidine (6-BG),bis-chloronitrosourea (BCNU) and camptothecin, or a therapeuticderivative of any thereof. Additional examples of therapeutic agentsthat may be suitable for use in accordance with the disclosed methodsinclude, without limitation, anti-restenosis, pro- oranti-proliferative, anti-inflammatory, anti-neoplastic, antimitotic,anti-platelet, anticoagulant, antifibrin, antithrombin, cytostatic,antibiotic and other anti-infective agents, anti-enzymatic,anti-metabolic, angiogenic, cytoprotective, angiotensin convertingenzyme (ACE) inhibiting, angiotensin II receptor antagonizing and/orcardioprotective agents. “Therapeutic agents” also refer to salts,acids, and free based forms of the above agents.

As used herein, the term “chemotherapeutic agent” when used in relationto cancer therapy, refers to any agent that results in the death ofcancer cells or inhibits the growth or spread of cancer cells. Examplesof such chemotherapeutic agents include alkylating agents, antibiotics,antimetabolitic agents, plant-derived agents, and hormones. In someembodiments, the chemotherapeutic agent is cisplatin. In someembodiments, the chemotherapeutic agent is oxaliplatin. In otherembodiments, the chemotherapeutic agent is gemcitabine. In otherembodiments, the chemotherapeutic agent is doxorubicin.

The term “antimetabolite” is used herein to refer to a therapeutic agentthat inhibits the utilization of a metabolite or a prodrug thereof.Examples of antimetabolites include methotrexate, pemetrexed,5-fluorouracil, 5-fluorouracil prodrugs such as capecitabine,5-fluorodeoxyuridine monophosphate, cytarabine, cytarabine prodrugs suchas nelarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine,azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, andcladribine. Anti-metabolites useful for practicing the disclosed methodsinclude nucleoside analogs, including a purine or pyrimidine analogs. Insome embodiments, platinum based chemotherapeutic agents are used incombination with an antimetabolite selection from the group consistingof fluoropyrimidine 5-fluorouracil, 5-fluoro-2′-deoxycytidine,cytarabine, gemcitabine, troxacitabine, decitabine, Azacytidine,pseudoisocytidine, Zebularine, Ancitabine, Fazarabine, 6- azacytidine,capecitabine, N⁴-octadecyl-cytarabine, elaidic acid cytarabine,fludarabine, cladribine, clofarabine, nelarabine, forodesine, andpentostatin, or a derivative thereof. In one example, the nucleosideanalog is a substrate for a nucleoside deaminase that is adenosinedeaminase or cytidine deaminase. In some examples, the nucleoside analogis selected from among fludarabine, cytarabine, gemcitabine, decitabineand azacytidine or a derivative thereof. In certain embodiments, theantimetabolite is 5-fluorouracil.

As used herein, a “taxane” or a “taxane-based agent” is an anti-canceragent that interferes with or disrupts microtubule stability, formationand/or function. Taxane agents include paclitaxel and docetaxel as wellas derivatives thereof, wherein the derivatives function againstmicrotubules by the same mode of action as the taxane from which theyare derived. In certain embodiments, the taxane is paclitaxel ordocetaxel, or a pharmaceutically acceptable salt, acid, or derivative ofpaclitaxel or docetaxel. In certain embodiments, the taxane ispaclitaxel (TAXOL®), docetaxel (TAXOTERE®), albumin-bound paclitaxel(nab-paclitaxel; ABRAXANE®), DHA-paclitaxel, or PG-paclitaxel.

The term “carrier” denotes an organic or inorganic ingredient, naturalor synthetic, with which liposome compositions are combined tofacilitate administration. The components of the pharmaceuticalcompositions are comingled in a manner that precludes interaction thatwould substantially impair their desired pharmaceutical efficiency.Suitable buffering agents include acetic acid and a salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V);and phosphoric acid and a salt (0.8-2% W/V). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9%W/V); and parabens (0.01-0.25% W/V).

The term “pharmaceutically-acceptable carrier” and “pharmaceuticallyacceptable carrier” refers to an ingredient in a pharmaceuticalformulation, other than an active ingredient, which is nontoxic to asubject. A pharmaceutically acceptable carrier includes, but is notlimited to, a buffer, carrier, excipient, stabilizer, diluent, orpreservative. Pharmaceutically-acceptable carriers can include forexample, one or more compatible solid or liquid filler, diluents orencapsulating substances which are suitable for administration to ahuman or other subject.

The terms “platinum-based chemotherapeutic agent”, “platinum-basedagent”, “platinum” and “platin”, are used interchangeably herein torefer to an antineoplastic drug that is a coordination complex ofplatinum. Examples of platinum-based chemotherapeutic agents includewithout limitation, cisplatin, oxaliplatin, nedaplatin, heptaplatin,lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin,dexormaplatin, spiroplatin picoplatin, triplatin, iproplatin, andoxaliplatin, zeniplatin, platinum-triamine complex; ormaplatin,dedaplatin, JM-216, 254-S, NK 121, CI-973, DWA 2114R, and NDDP.

The term “platinum-based chemotherapy” refers to therapy with one ormore platinum-based chemotherapeutic agents, optionally in combinationwith one or more other therapeutic agents (e.g., chemotherapeuticagents, such as gemcitabine, doxorubicin, an antifolate, and a taxane).

In one embodiment, the disclosure provides a liposome compositioncomprising a liposome encapsulating a complex of a platinum-basedchemotherapeutic agent or a salt thereof, and one or more polyglutamatemolecules. In some embodiments, the liposome is pegylated. In someembodiments, the liposome composition comprises on ore morepharmaceutically acceptable carriers.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of cisplatin or a salt thereof, and one or morepolyglutamate molecules; and one or more pharmaceutically acceptablecarriers. In further embodiments, the liposome is pegylated.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of a cisplatin analog or a salt thereof, and oneor more polyglutamate molecules.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of carboplatin or a salt thereof, and one ormore polyglutamate molecules. In some embodiments, the liposomecomposition comprises a liposome encapsulating a complex of a complex ofoxaliplatin or a salt thereof, and one or more polyglutamate molecules.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of a platinum-based chemotherapeutic agent andone or more polyglutamate molecules, wherein the platinum-basedchemotherapeutic is selected from the group: nedaplatin, heptaplatin,and lobaplatin, or a salt thereof, and cyclodextrin; and one or morepharmaceutically acceptable carriers. In some embodiments, theplatinum-based chemotherapeutic agent is nedaplatin. In someembodiments, the platinum-based chemotherapeutic agent is heptaplatin.In some embodiments, the platinum-based chemotherapeutic agent islobaplatin.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of a platinum-based chemotherapeutic agent andone or more polyglutamate molecules, wherein the platinum-basedchemotherapeutic is selected from the group: stratoplatin, paraplatin,platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin,triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin,platinum-triamine, enloplatin, JM-216, 254-S, NK 121, CI-973, DWA 2114R,NDDP, and dedaplatin. In some embodiments, the platinum-basedchemotherapeutic agent is stratoplatin. In some embodiments, theplatinum-based chemotherapeutic agent is paraplatin. In someembodiments, the platinum-based chemotherapeutic agent is platinol. Insome embodiments, the platinum-based chemotherapeutic agent iscycloplatin. In some embodiments, the platinum-based chemotherapeuticagent is dexormaplatin. In some embodiments, the platinum-basedchemotherapeutic agent is spiroplatin. In some embodiments, theplatinum-based chemotherapeutic agent is picoplatin. In someembodiments, the platinum-based chemotherapeutic agent is triplatin. Insome embodiments, the platinum-based chemotherapeutic agent istetraplatin. In some embodiments, the platinum-based chemotherapeuticagent is iproplatin. In some embodiments, the platinum-basedchemotherapeutic agent is ormaplatin. In some embodiments, theplatinum-based chemotherapeutic agent is zeniplatin. In someembodiments, the platinum-based chemotherapeutic agent is picoplatin. Insome embodiments, the platinum-based chemotherapeutic agent istriplatin. In some embodiments, the platinum-based chemotherapeuticagent is tetraplatin. In some embodiments, the platinum-basedchemotherapeutic agent is iproplatin. In some embodiments, theplatinum-based chemotherapeutic agent is ormaplatin. In someembodiments, the platinum-based chemotherapeutic agent is zeniplatin. Insome embodiments, the platinum-based chemotherapeutic agent isplatinum-triamine. In some embodiments, the platinum-basedchemotherapeutic agent is enloplatin. In some embodiments, theplatinum-based chemotherapeutic agent is JM-216. In some embodiments,the platinum-based chemotherapeutic agent is 254-S. In some embodiments,the platinum-based chemotherapeutic agent is NK 121. In someembodiments, the platinum-based chemotherapeutic agent is CI-973. Insome embodiments, the platinum-based chemotherapeutic agent is DWA2114R. In some embodiments, the platinum-based chemotherapeutic agent isNDDP. In some embodiments, the platinum-based chemotherapeutic agent isdedaplatin.

In another embodiment, the disclosure provides a liposome compositioncomprising a liposome encapsulating a complex of a platinum-basedchemotherapeutic agent or a salt thereof, and a cyclodextrin. In someembodiments, the liposome is pegylated. In some embodiments, theliposome composition comprises on ore more pharmaceutically acceptablecarriers.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of cisplatin or a salt thereof, and acyclodextrin.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of a cisplatin analog or a salt thereof, and acyclodextrin.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of carboplatin or a salt thereof, and acyclodextrin

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of a complex of oxaliplatin or a salt thereof,and a cyclodextrin.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of a platinum-based chemotherapeutic agent and acyclodextrin, wherein the platinum-based chemotherapeutic agent isselected from the group: nedaplatin, heptaplatin, and lobaplatin, or asalt thereof, and cyclodextrin; and one or more pharmaceuticallyacceptable carriers. In some embodiments, the platinum-basedchemotherapeutic agent is nedaplatin. In some embodiments, theplatinum-based chemotherapeutic agent is heptaplatin. In someembodiments, the platinum-based chemotherapeutic agent is lobaplatin.

In some embodiments, the liposome composition comprises a liposomeencapsulating a complex of a platinum-based chemotherapeutic agent and acyclodextrin, wherein the platinum-based chemotherapeutic agent isselected from the group: stratoplatin, paraplatin, platinol,cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin,tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine,enloplatin, JM-216, 254-S, NK 121, CI-973, DWA 2114R, NDDP, anddedaplatin. In some embodiments, the platinum-based chemotherapeuticagent is stratoplatin. In some embodiments, the platinum-basedchemotherapeutic agent is paraplatin. In some embodiments, theplatinum-based chemotherapeutic agent is platinol. In some embodiments,the platinum-based chemotherapeutic agent is cycloplatin. In someembodiments, the platinum-based chemotherapeutic agent is dexormaplatin.In some embodiments, the platinum-based chemotherapeutic agent isspiroplatin. In some embodiments, the platinum-based chemotherapeuticagent is picoplatin. In some embodiments, the platinum-basedchemotherapeutic agent is triplatin. In some embodiments, theplatinum-based chemotherapeutic agent is tetraplatin. In someembodiments, the platinum-based chemotherapeutic agent is iproplatin. Insome embodiments, the platinum-based chemotherapeutic agent isormaplatin. In some embodiments, the platinum-based chemotherapeuticagent is zeniplatin. In some embodiments, the platinum-basedchemotherapeutic agent is picoplatin. In some embodiments, theplatinum-based chemotherapeutic agent is triplatin. In some embodiments,the platinum-based chemotherapeutic agent is tetraplatin. In someembodiments, the platinum-based chemotherapeutic agent is iproplatin. Insome embodiments, the platinum-based chemotherapeutic agent isormaplatin. In some embodiments, the platinum-based chemotherapeuticagent is zeniplatin. In some embodiments, the platinum-basedchemotherapeutic agent is platinum-triamine. In some embodiments, theplatinum-based chemotherapeutic agent is enloplatin. In someembodiments, the platinum-based chemotherapeutic agent is JM-216. Insome embodiments, the platinum-based chemotherapeutic agent is 254-S. Insome embodiments, the platinum-based chemotherapeutic agent is NK 121.In some embodiments, the platinum-based chemotherapeutic agent isCI-973. In some embodiments, the platinum-based chemotherapeutic agentis DWA 2114R. In some embodiments, the platinum-based chemotherapeuticagent is NDDP. In some embodiments, the platinum-based chemotherapeuticagent is dedaplatin. In further embodiments, the liposome is pegylated.

Cisplatin is a potent anti-carcinogen that is widely used for varioussolid tumors; however, its clinical application is limited by its severenephrotoxicity. It has been widely used because of its potent cytotoxiceffects upon a variety of tumor types including testicular, ovarian, andcervical carcinoma. In some embodiments, the disclosure provides amethod for treating cancer in a subject, comprising administering aneffective amount of a liposome composition comprising a liposomeencapsulating a complex of a platinum-based chemotherapeutic agent or asalt thereof, and (a) one or more polyglutamate molecules, or (b)cyclodextrin; to a subject having or at risk of having cancer. In someembodiments the platinum-based chemotherapeutic agent is cisplatin. Insome embodiments the platinum-based chemotherapeutic agent is cisplatina cispatin analoag. In some embodiments the cancer is a member selectedfrom the group: lung cancer, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma; and a hematologicmalignancy such as for example, a leukemia, a lymphoma and other B cellmalignancies, myeloma and other plasma cell dyscrasias. In furtherembodiments, the cancer is testicular cancer, ovarian cancer, orcervical carcinoma.

In some embodiments, the disclosure provides compositions such asliposome compositions, that comprise a complex of a therapeutic agentand a cyclodextrin. Cyclodextrins (CDs) are groups of cyclicoligosaccharides which have been shown to improve physicochemicalproperties of many drugs through formation of complexes. CDs are cyclicoligosaccharides composed of several D-glucose units linked by a-(1, 4)bonds. This cyclic configuration provides a hydrophobic internal cavityand gives the CDs a truncated cone shape. Many hydroxyl groups aresituated on the edges of the ring which make the CDs both lipophilic andsoluble in water. As a result, CDs are able to form complexes with awide variety of hydrophobic agents, and thus change thephysical-chemical properties of these complexed agents.

The terms “cyclodextrin” or “CD” unless otherwise specified herein,refer generally to a parent or derivatized cyclic oligosaccharidecontaining a variable number of (α-1,4)-linked D-glucopyranoside unitsthat is able to form a complex with a platinum-based agent, or anotherchemotherapeutic agent of interest (e.g., an antifolate, gemcitabine,and doxorubicin). Each cyclodextrin glucopyranoside subunit hassecondary hydroxyl groups at the 2 and 3 positions and a primaryhydroxyl group at the 6-position. The terms “parent,” “underivatized,”or “inert,” cyclodextrin refer to a cyclodextrin containingD-glucopyranoside units having the basic formula C₆H₁₂O₆ and a glucosestructure without any additional chemical substitutions (e.g.,α-cyclodextrin consisting of 6 D-glucopyranoside units, a β-cyclodextrinconsisting of 7 D-glucopyranoside units, and a γ-cyclodextrin consistingof 8 D-glucopyranoside units). The physical and chemical properties of aparent cyclodextrin can be modified by derivatizing the hydroxyl groupswith other functional groups. Any substance located within thecyclodextrin internal phase is said to be “complexed” with thecyclodextrin, or to have formed a complex (inclusion complex) with thecyclodextrin.

As used herein, there are no particular limitations on the cyclodextrincontained in the provided liposome compositions so long as thecyclodextrins can encapsulate a desired therapeutic agent, In particularembodiments, the cyclodextrins have been derivatized to bear ionizable(e.g., weakly basic and/or weakly acidic) functional groups tofacilitate encapsulation by the liposomes.

Modifications of the hydroxyl groups of cyclodextrins, such as thosefacing away from the cyclodextrin interior phase, with ionizablechemical groups is known to facilitate the loading of cyclodextrins andtherapeutic agents complexed with the cyclodextrins. In someembodiments, the cyclodextrins in the provided liposome compositionshave at least 2, 3, 4, 5, 6, 6, 7, 8, 9, or 10 hydroxyl groupsubstituted with an ionizable chemical group. The term “chargedcyclodextrin” refers to a cyclodextrin having one or more of itshydroxyl groups substituted with a charged moiety. Such a moiety canitself be a charged group or it can comprise an organic moiety (e.g., aC₁-C₆ alkyl or C₁-C₆ alkyl ether moiety) substituted with one or morecharged moieties.

In some embodiments, the “ionizable” or “charged” moieties of a CDderivative are weakly ionizable. Weakly ionizable moieties are thosethat are either weakly basic or weakly acidic. Weakly basic functionalgroups (W) have a pKa of between about 6.0-9.0, 6.5-8.5, 7.0-8.0,7.5-8.0, and any range in between inclusive according to CH3-W.Similarly, weakly acidic functional groups (X) have a log dissociationconstant (pKa) of between about 3.0-7.0, 4.0-6.5, 4.5-6.5, 5.0-6.0,5.0-5.5, and any range in between inclusive according to CH3-X.Representative anionic moieties include, without limitation,carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkylether, sulphate carbonate, thiocarbonate, dithiocarbonate, phosphate,phosphonate, sulfonate, nitrate, and borate groups. Representativecationic moieties include, without limitation, amino, guanidine, andquarternary ammonium groups.

In another embodiment, the derivatized cyclodextrin is a “polyanion” or“polycation.” A polyanion is a derivatized cyclodextrin having more thanone negatively charged group resulting in net a negative ionic charge ofmore than two units. A polycation is a derivatized cyclodextrin havingmore than one positively charged group resulting in net positive ioniccharger of more than two units.

In another embodiment, the derivatized cyclodextrin is a “chargeableamphiphile.” By “chargeable” is meant that the amphiphile has a pK inthe range pH 4 to pH 8 or 8.5. A chargeable amphiphile may therefore bea weak acid or base. By “amphoteric” herein is meant a derivatizedcyclodextrin having a ionizable groups of both anionic and cationiccharacter wherein: (a) at least one, and optionally both, of the cationand anionic amphiphiles is chargeable, having at least one charged groupwith a pK between 4 and 8 to 8.5, (b) the cationic charge prevails at pH4, and (c) the anionic charge prevails at pH 8 to 8.5.

In some embodiments, the “ionizable” or “charged” derivatizedcyclodextrin as a whole, whether polyionic, amphiphilic, or otherwise,are weakly ionizable (i.e., have a pKai of between about 4.0-8.5,4.5-8.0, 5.0-7.5, 5.5-7.0, 6.0-6.5, and any range in between inclusive).

Any one, some, or all hydroxyl groups of any one, some or allα-D-glucopyranoside units of a cyclodextrin can be modified to anionizable chemical group as provided herein. Since each cyclodextrinhydroxyl group differs in chemical reactivity, reaction with a modifyingmoiety can produce an amorphous mixture of positional and opticalisomers. Alternatively, certain chemistry can allow for pre-modifiedα-D-glucopyranoside units to be reacted to form uniform products.

The aggregate substitution that occurs for cyclodextrin derivatives in amixture is described by a term referred to as the degree ofsubstitution. For example, a 6-ethylenediamino-β-cyclodextrin with adegree of substitution of seven would be composed of a distribution ofisomers of 6-ethylenediamino-β-cyclodextrin in which the average numberof ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin moleculeis seven. The degree of substitution for a cyclodextrin derivativemixture can routinely be determined using mass spectrometry or nuclearmagnetic resonance spectroscopy.

In one embodiment, at least one hydroxyl moiety facing away from thecyclodextrin interior is substituted with an ionizable chemical group.For example, the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and allthree of C2-C3-C6 hydroxyls of at least one α-D-glucopyranoside unit aresubstituted with an ionizable chemical group. Any such combination ofhydroxyls can similarly be combined with at least two, three, four,five, six, seven, eight, nine, ten, eleven, up to all of thealpha-D-glucopyranoside units in the modified cyclodextrin as well as incombination with any degree of substitution provided herein. One suchderivative is a sulfoalkyl ether cyclodextrin (SAE-CD). Sulfobutyl etherderivatives of beta cyclodextrin (SBE-β-CD) have been demonstrated tohave significantly improved aqueous solubility compared to the parentcyclodextrin.

Additional cyclodextrin derivatives that may be complexed withtherapeutic agents in the provided liposome compositions includesugammadex or Org-25969, in which the 6-hydroxy groups on γ-CD have beenreplaced by carboxythio acetate ether linkages, and hydroxybutenyl-β-CD.Alternative forms of cyclodextrin include: 2,6-Di-O-methyl-β-CD (DIMEB),2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), randomlymethylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin(SBE-β-CD), and sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylatedbeta-cyclodextrin sodium salt, sulfobutylated beta-cyclodextrin sodiumsalt, (2-Hydroxypropyl)-alpha-cyclodextrin,(2-Hydroxypropyl)-beta-cyclodextrin, (2-Hydroxypropyl)-γ-cyclodextrin,2,6-di-O-methyl)-beta-cyclodextrin (DIMEB-50 Heptakis),2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB Heptakis),methyl-beta-cyclodextrin, octakis (6-deoxy-6-iodo)-γ-cyclodexrin, and,octakis (6-deoxy-6-bromo)-gamma-cyclodexrin.

In some embodiments, the cyclodextrin(s) has a high solubility in waterin order to facilitate entrapment of a larger amount of the cyclodextrinin the liposome internal phase. In some embodiments, the watersolubility of the cyclodextrin is at least 10 mg/mL, 20 mg/mL, 30 mg/mL,40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL orhigher. In some embodiments, the water solubility of the cyclodextrin(s)is within a range of 10-150 mg/mL, 20-100 mg/mL 20-75 mg/mL, and anyrange in between inclusive.

In some embodiments, a large association constant between thecyclodextrin and the therapeutic agent is preferable and can be obtainedby selecting the number of glucose units in the cyclodextrin based onthe size of the therapeutic agent (see, for example, Albers et al.,Crit. Rev. Therap. Drug Carrier Syst. 12:311-337 (1995); Stella et al.,Toxicol. Pathol. 36:30-42 (2008). When the association constant dependson pH, the cyclodextrin can be selected such that the associationconstant becomes large at the pH of the liposome internal phase. As aresult, the solubility (nominal solubility) of the therapeutic agent inthe presence of cyclodextrin can be further improved. In someembodiments, the association constant of the cyclodextrin with thetherapeutic agent is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000,or higher. In some embodiments, the association constant of thecyclodextrin with the therapeutic agent is in the range 100-1,200,200-1,000, 300-750, and any range in between inclusive.

In some embodiments, the cyclodextrin of the pharmaceutical compositionis underivatized.

In some embodiments, the cyclodextrin derivative of the pharmaceuticalcomposition has the structure of Formula I:

wherein: n is 4, 5, or 6;wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each, independently,—H, a straight chain or branched C₁-C₈- alkylene group, or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈- alkylene (e.g., C₁-C₈-(alkylene)-SO₃ ⁻ group);

In some embodiments, the cyclodextrin derivative of the liposomecomposition has the structure of formula II:

wherein: n is 4, 5, or 6;

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each, independently,—O— or a —O—(C₂-C₆ alkylene)-SO₃— group; wherein at least one of R₁ andR₂ is independently a —O—(C₂-C₆ alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃,S₄, S₅, S₆, S₇, S₈, and S₉ are each, independently, a pharmaceuticallyacceptable cation. In further embodiments, the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca+2, or Mg+2, and ammonium ions andamine cations such as the cations of (C1-C6)-alkylamines, piperidine,pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine. In someembodiments, at least one of R1 and R2 is independently a —O—(C2-C6alkylene)-SO3- group that is a —O—(CH₂)_(m)SO₃— group, wherein m is 2 to6, preferably 2 to 4, (e.g., —O—CH2CH2CH2S03- or —O—CH2CH2CH2CH2S03-);and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each, independently, H ora pharmaceutically cation which includes for example, alkali metals(e.g., Li⁺, Na⁺, K⁺) alkaline earth metals (e.g., Ca⁺², Mg⁺²), ammoniumions and amine cations such as the cations of (C1-C6)-alkylamines,piperidine, pyrazine, (C1-C6)-alkanolamine and(C4-C8)-cycloalkanolamine:

In some embodiments, a cyclodextrin derivative of the liposomecomposition is a cyclodextrin disclosed in U.S. Pat. Nos. 6,133,248,5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; andIntl. Appl. Publ. No. WO 02005/117911, the contents each of which isherein incorporated by reference in its priority.

In some embodiments, the cyclodextrin derivative of the liposomecomposition is a sulfoalkyl ether cyclodextrin. In some embodiments, thecyclodextrin derivative of the liposome composition is a sulfobutylether-3-cyclodextrin such as CAPTISOL® (CyDex Pharma Inc., Lenexa,Kans.). Methods for preparing sulfobutyl ether-3- cyclodextrin and othersulfoalkyl ether cyclodextrins are known in the art.

In some embodiments, the cyclodextrin derivative in the liposomecomposition is a compound of Formula III:

wherein R equals:

-   -   (a) (H)_(21-X) or (—(CH₂)₄—SO₃Na)_(x), and x=1.0-10.0, 1.0-5.0,        6.0-7.0 or 8.0-10.0;    -   (b) (H)_(21-X) or (—(CH₂CH(OH)CH₃)_(x), and x=1.0-10.0, 1.0-5.0,        6.0-7.0 or 8.0-10.0;    -   (c) (H)_(21-X) or (sulfoalkyl ethers)_(x), and x=1.0-10.0,        1.0-5.0, 6.0-7.0 or 8.0-10.0; or    -   (d) (H)_(21-X) or (—(CH₂)₄—SO₃Na)_(x), and x=1.0-10.0, 1.0-5.0,        6.0-7.0 or 8.0-10.0.

In some embodiments, the provided compositions comprise liposomedelivery vehicles. Liposomes are phospholipid vesicles composed of lipidbilayers enclosing one or more aqueous compartments. The term “liposome”refers to a microscopic closed vesicle having an internal phase enclosedby a lipid bilayer. A liposome can be a small single-lipid bilayerliposome such as a small unilamellar vessicle (SUV), largesingle-membrane liposome such as a large unilamellar vesicle (LUV), astill larger single-membrane liposome such as a giant unilamellarvesicle (GUV), a multilayer liposome having multiple concentric lipidbilayers such as a multilamellar vesicle (MLV), or a liposome havingmultiple lipid bilayers that are irregular and not concentric such as amultivesicular vesicle (MVV). See, e.g., U.S. Pat. Publ. 2012/0128757;U.S. Pat. Nos. 4,235,871 and 4,737,323; and Intl. Appl. Publ. No. WO96/14057.

The term “liposome composition” as used herein, refers to a compositioncomprising liposomes that in turn contain complexes of a therapeuticagent and one or more polyglutamate molecules or cyclodextrin in theinternal phase of the liposome. Liposome compositions can include solidand liquid forms. In instances where the liposome composition is in asolid form, the liposome composition can be made into a liquid form bydissolving or suspending it in a pharmaceutically acceptable solvent. Inthe case where the liposome composition is frozen solid, the compositioncan be made into a liquid form by melting.

The concentration of liposome and the concentration of the therapeuticagent in the liposome composition can be appropriately set according tothe liposome composition objective, formulation, and otherconsiderations known to the skilled artisan.

In some embodiments, the liposomes in the liposome composition aretargeted pegylated liposomes. In some embodiments, the pegylatedliposomes contain complexes of platinum-based chemotherapeutic agentsand cyclodextrin within their aqueous internal phase, a PEG moleculeattached to an exterior of the liposome; and a targeting moietycomprising a protein (e.g., an antibody or antibody fragment) withspecific affinity for at least one antigen expressed on the surface ofthe cancer cell (e.g., a folate receptor such as FR-α, FR-β and/orFR-δ)), and wherein the targeting moiety is attached to at least one ofthe PEG and the exterior of the liposome. In other embodiments, thepegylated liposomes contain complexes of platinum-based chemotherapeuticagents and polyglutamate molecules within their aqueous internal phase,a PEG molecule attached to an exterior of the liposome; and a targetingmoiety comprising a protein (e.g., an antibody or antibody fragment)with specific affinity for at least one antigen expressed on the surfaceof the cancer cell (e.g., a folate receptor such as FR-α, FR-β and/orFR-δ)), and wherein the targeting moiety is attached to at least one ofthe PEG and the exterior of the liposome.

The lipids and other components of the liposomes contained in theliposome compositions can be any lipid, lipid combination and ratio, orcombination of lipids and other liposome components and their respectiveratios known in the art. However, it will be understood by one skilledin the art that liposomal encapsulation of any particular drug, such as,and without limitation, therapeutic agents such as platinum-basedchemotherapeutic agents discussed herein, may involve substantialroutine experimentation to achieve a useful and functional liposomalformulation. However, it will be understood by one skilled in the artthat liposomal encapsulation of any particular drug, such as, andwithout limitation, complexes of platinum-based chemotherapeutic agentsand one or more polyglutamate molecules, complexes of platinum-basedchemotherapeutic agents and cyclodextrin, complexes of gemcitabine-basedchemotherapeutic agents and one or more polyglutamate molecules orcyclodextrin, and complexes of polyglutamated antifolate-basedchemotherapeutic agents and cyclodextrin. In general, the providedliposomes may have any liposome structure, e.g., structures having aninner space sequestered from the outer medium by one or more lipidbilayers, or any microcapsule that has a semi-permeable membrane with alipophilic central part where the membrane sequesters an interior. Thelipid bilayer can be any arrangement of amphiphilic moleculescharacterized by a hydrophilic part (hydrophilic moiety) and ahydrophobic part (hydrophobic moiety). Usually amphiphilic molecules ina bilayer are arranged into two dimensional sheets in which hydrophobicmoieties are oriented inward the sheet while hydrophilic moieties areoriented outward. Amphiphilic molecules forming the provided liposomescan be any known or later discovered amphiphilic molecules, e.g., lipidsof synthetic or natural origin or biocompatible lipids. The liposomescan also be formed by amphiphilic polymers and surfactants, e.g.,polymerosomes and niosomes. For the purpose of this disclosure, withoutlimitation, these liposome-forming materials also are referred to as“lipids”.

The formulations provided herein such as liposome compositionformulations, can be in liquid or dry form such as a dry powder or drycake. The dry powder or dry cake may have undergone primary dryingunder, for example, lyophilization conditions or optionally, the drycake or dry powder may have undergone both primary drying only or bothprimary drying and secondary drying. In the dry form, the powder or cakemay, for example, have between 1% to 6% moisture, for example, such asbetween 2% to 5% moisture or between 2% to 4% moisture. One examplemethod of drying is lyophilization (also called freeze-drying, orcyrodessication). Any of the compositions and methods of the disclosuremay include liposomes, lyophilized liposomes or liposomes reconstitutedfrom lyophilized liposomes. In some embodiments, the disclosedcompositions and methods include one or more lyoprotectants orcryoprotectants. These protectants are typically polyhydroxy compoundssuch as sugars (mono-, di-, and polysaccharides), polyalcohols, andtheir derivatives, glycerol, or polyethyleneglycol, trehalose, maltose,sucrose, glucose, lactose, dextran, glycerol, or aminoglycosides. Infurther embodiments, the lyoprotectants or cryoprotectants comprise upto 10% or up to 20% of a solution outside the liposome, inside theliposome, or both outside and inside the liposome.

In some embodiments, the liposomes include a steric stabilizer thatincreases their longevity in circulation. One or more steric stabilizerssuch as a hydrophilic polymer (Polyethylene glycol (PEG)), a glycolipid(monosialoganglioside (GM1)) or others occupies the space immediatelyadjacent to the liposome surface and excludes other macromolecules fromthis space. Consequently, access and binding of blood plasma opsonins tothe liposome surface are hindered, and thus interactions of macrophageswith such liposomes, or any other clearing mechanism, are inhibited andlongevity of the liposome in circulation is enhanced. In someembodiments, the steric stabilizer or the population of stericstabilizers is a PEG or a combination comprising PEG. In furtherembodiments, the steric stabilizer is a PEG or a combination comprisingPEG with a number average molecular weight (Mn) of 200 to 5000 daltons.These PEG(s) can be of any structure such as linear, branched, star orcomb structure and are commercially available.

The diameter of the liposomes is not particularly limited. Depending onthe desired application, the particle size of the liposome can beregulated. For example, when it is intended to transmit liposome tocancerous tissue or inflamed tissue by the Enhanced Permeability andRetention (EPR) effect as an injection product or the like, it ispreferable that liposome particle size be 30-400 nm, 50-200 nm, 75-100nm, and any range in between. In the case where the intention is totransmit liposome to macrophage, it is preferable that liposome particlesize be 30 to 1000 nm, and it is more preferable that the particle sizebe 100 to 400 nm. It should be noted that in normal tissue, vascularwalls serve as barriers (because the vascular walls are denselyconstituted by vascular endothelial cells), and microparticles such assupermolecules and liposome of specified size cannot be distributedwithin the tissue. However, in diseased tissue, vascular walls are loose(because interstices exist between vascular endothelial cells),increasing vascular permeability, and supermolecules and microparticlescan be distributed to extravascular tissue (enhanced permeability).Moreover, the lymphatic system is well developed in normal tissue, butit is known that the lymphatic system is not developed in diseasedtissue, and that supermolecules or microparticles, once incorporated,are not recycled through the general system, and are retained in thediseased tissue (enhanced retention), which forms the basis of the EPReffect. Thus, it is possible to control liposome pharmacokinetics byadjusting liposome particle size.

In some embodiments, the liposomes have a diameter in the range of forexample, 10-250 nm. In some embodiments, the liposomes have a diameterin the range of for example, 30-150 nm. In other embodiments, theliposomes have a diameter in the range of 40-70 nm.

The properties of liposomes are influenced by the nature of lipids usedto make the liposomes. A wide variety of lipids have been used to makeliposomes. These include cationic, anionic and neutral lipids. In someembodiments, the liposomes comprising the complexes containingcyclodextrin and the therapeutic agent (e.g., a platinum-basedchemotherapeutic agent, a doxorubicin-based chemotherapeutic agent, agemcitabine-based chemotherapeutic agent, and a taxane-basedchemotherapeutic agent) are anionic or neutral. In other embodiments,the provided liposomes are cationic. The determination of the charge(e.g., anionic, neutral or cationic) can routinely be determined bymeasuring the zeta potential of the liposome. The zeta potential of theliposome can be positive, zero or negative. In some embodiments, thezeta potential of the liposome is less than or equal to zero. In someembodiments, the zeta potential of the liposome is in a range of 0 to−150 mV. In another embodiment, the zeta potential of the liposome is inthe range of −30 to −50 mV.

Alternatively, in some embodiments, the zeta potential of the liposomeis greater than zero. In some embodiments, the zeta potential of theliposome between 1 and 100 mV. In another embodiment, the zeta potentialof the liposome is in the range of 5 to 60 mV. In another embodiment,the zeta potential of the liposome is in the range of 10 to 50 mV.Techniques and reagents are known in the art for routinely determiningthe zeta potential of a liposome such as, dynamic light scattering usinga Malvern Zetasizer ZS).

In some embodiments, cationic lipids are used to make cationic liposomeswhich are commonly used as gene transfection agents. The positive chargeon cationic liposomes enables interaction with the negative charge oncell surfaces. Following binding of the cationic liposomes to the cell,the liposome is transported inside the cell through endocytosis.

In some preferred embodiments, a neutral to anionic liposome is used. Ina preferred embodiment, an anionic liposome is used. Using a mixture of,for example, neutral lipids such as HSPC and anionic lipids such asPEG-DSPE results in the formation of anionic liposomes which are lesslikely to non-specifically bind to normal cells. Specific binding totumor cells can be achieved by using a tumor targeting antibody such as,for example, a folate receptor antibody, including, for example, folatereceptor alpha antibody, folate receptor beta antibody and/or folatereceptor delta antibody.

As an example, at least one (or some) of the lipids is/are amphipathiclipids, defined as having a hydrophilic and a hydrophobic portions(typically a hydrophilic head and a hydrophobic tail). The hydrophobicportion typically orients into a hydrophobic phase (e.g., within thebilayer), while the hydrophilic portion typically orients toward theaqueous phase (e.g., outside the bilayer). The hydrophilic portion cancomprise polar or charged groups such as carbohydrates, phosphate,carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other likegroups. The hydrophobic portion can comprise apolar groups that includewithout limitation long chain saturated and unsaturated aliphatichydrocarbon groups and groups substituted by one or more aromatic,cyclo-aliphatic or heterocyclic group(s). Examples of amphipathiccompounds include, but are not limited to, phospholipids, aminolipidsand sphingolipids.

Typically, the lipids making up the liposomes in the provided liposomecompositions are phospholipids. Phospholipids include without limitationphosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidylinositol, phosphatidylserine, and the like. It is to beunderstood that other lipid membrane components, such as cholesterol,sphingomyelin, and cardiolipin, can be used.

The lipid bilayer of the liposome comprises phospholipids and/orphospholipid derivatives. In addition to phospholipids and/orphospholipid derivatives, the liposome can further include sterols, suchas cholesterol and cholestanol as membrane stabilizers and fatty acidshaving saturated or unsaturated acyl groups, such as those having acarbon number of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, orlonger. Representative, non-limiting examples include acyl groupsderived from fatty-acid such as lauric acid, myristic acid, palmiticacid, stearic acid, oleic acid, and linoleic acid.

In some embodiments, the lipid bilayer comprises a phospholipid. In someembodiments, the lipid bilayer comprises one or more differentphospholipids. In additional embodiments, the lipid bilayer comprises aphospholipid derived from a natural substance. In further embodiments,the lipid bilayer comprises a phospholipid derived from a memberselected from the group: egg-yolk lecithin and soy lecithin, partiallyhydrogenated egg-yolk lecithin, (completely) hydrogenated egg-yolklecithin, partially hydrogenated soy lecithin, and (completely)hydrogenated soy lecithin whose unsaturated fatty-acid residues arepartially or completely hydrogenated.

In some embodiments, the lipid bilayer comprises a phospholipidderivative. In some embodiments, the lipid bilayer comprises one or moredifferent phospholipid derivatives.

In some embodiments, the lipid bilayer comprises both phospholipids andphospholipid derivatives.

In some embodiments, the lipid bilayer comprises one or more differentphospholipids and one or more different phospholipid derivatives. Insome embodiments, the lipid bilayer comprises a phospholipid or aphospholipid derivative selected from the group: phosphatidylethanolamine, phosphatidyl choline, phosphatidyl serine, phosphatidylinositol, phosphatidyl glycerol, cardiolipin, sphingomyelin, ceramidephosphorylethanolamine, ceramide phosphoryl glycerol, ceramidephosphoryl glycerol phosphate, 1,2-dimyristoyl- 1,2-deoxyphosphatidylcholine, plasmalogen, and phosphatidic acid. It is also acceptable tocombine one or more of these phospholipids and phospholipid derivatives.

Representative, non-limiting examples of modified lipids that can beincluded in the provided liposome compositions include PEG lipids, sugarlipids, antibody-modified lipids, peptide-modified lipids, and the like.Liposomes containing such modified lipids can be targeted to desiredtarget cells or target tissue. Also, there are no particular limitationson the mixing amount (mole fraction) of functional lipids and modifiedlipids used when preparing the liposome. In some embodiments, suchlipids make up 0-50%, 0-40%, 0-30%, 0-20%, 0-15%, 0-10%, 0-5%, 0-1% orless of the entirety of liposome lipid bilayer constituent lipids.

The lipids comprising the liposomes in the provided liposomecompositions can be anionic and neutral (including zwitterionic andpolar) lipids including anionic and neutral phospholipids. Neutrallipids exist in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,dioleoyl-phosphatidylglycerol (DOPG), diacylphosphatidylcholine,diacylphosphatidylethanol-amine, ceramide, sphingomyelin, cephalin,cholesterol, cerebrosides and diacylglycerols. Examples of zwitterioniclipids include without limitation dioleoylphosphatidylcholine (DOPC),dimyristoylphospha-tidylcholine (DMPC), and dioleoylphosphatidylserine(DOPS). Anionic lipids are negatively charged at physiological pH. Theselipids include without limitation phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dode-canoylphosphatidylethanolamines, N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

Collectively, anionic and neutral lipids are referred to herein asnon-cationic lipids. Such lipids may contain phosphorus but they are notso limited. Examples of non-cationic lipids include lecithin,lysolecithin, phosphatidylethanolamine, lysophosphatidyl-ethanolamine,dioleoylphosphati-dylethanolamine (DOPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidy 1-ethanolamine (DSPE),palmitoyloleoyl-phosphatidylethanolamine (POPE)palmitoyl-oleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphat-idylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),palmitoyloleyolphosphatidylglycerol (POPG), 16-0-monomethyl PE,16-0-dimethyl PE, 18-1-trans PE,palmitoyloleoyl-phosphatidylethanolamine (POPE),1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), phosphatidylserine,phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetyl-phosphate, and cholesterol.

The liposomes may be assembled using any liposomal assembly method usingliposomal components (also referred to as liposome components) known inthe art. Liposomal components include, for example, lipids such as DSPE,HSPC, cholesterol and derivatives of these components. Other suitablelipids are commercially available for example, by Avanti Polar Lipids,Inc. (Alabaster, Ala., USA). A partial listing of available negativelyor neutrally charged lipids suitable for making anionic liposomes, canbe, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC,DOPC, DMPE, DPPE, DOPE, DMPA.Na, DPPA.Na, DOPA.Na, DMPG.Na, DPPG.Na,DOPG.Na, DMPS.Na, DPPS.Na, DOPS.Na, DOPE-Glutaryl.(Na)2, TetramyristoylCardiolipin.(Na)2, DSPE-mPEG-2000.Na, DSPE-mPEG-5000.Na, andDSPE-Maleimide PEG-2000.Na.

In some embodiments, the liposome compositions provided herein areformulated in a liposome comprising a cationic lipid. In one embodiment,the cationic lipid is selected from, but not limited to, a cationiclipid described in Intl. Appl. Publ. Nos. WO 2012/040184, WO2011/153120,WO2011/149733, WO2011/090965, WO2011/043913, WO2011/022460,WO2012/061259, WO2012/054365, WO2012/044638, WO 2010/080724,WO2010/21865 and WO2008/103276, U.S. Pat. Nos. 7,893,302, 7,404,969 and8,283,333 and US Appl. Publ. Nos. US20100036115 and US20120202871; eachof which is herein incorporated by reference in their entirety. Inanother embodiment, the cationic lipid may be selected from, but notlimited to, formula A described in Intl. Appl. Publ. Nos. WO2012/040184,WO2011/153120, WO2011/1149733, WO2011/090965, WO2011/043913,WO2011/022460, WO2012/061259, WO2012/054365 and WO2012/044638; each ofwhich is herein incorporated by reference in their entirety. In yetanother embodiment, the cationic lipid may be selected from, but notlimited to, formula CLI-CLXXIX of International Publication No.WO2008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formulaCLI-CLXXXXII of U.S. Pat. No. 7,404,969 and formula I-VI of USPublication No. US20100036115; each of which is herein incorporated byreference in their entirety. As a non-limiting example, the cationiclipid may be selected from(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemyl-hexa-cosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-16,19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethyl-henicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyl-tricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexa-cosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)—N,N-dimethyl-triaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl] pyrrolidine,(20Z)—N,N-dimethyl-heptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpenta-cos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylheni-cosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclo-propyl] eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethyl nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]-nonadecan-10-amine,N,N-dimethyl-21-[R1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecyl-cyclopropyl] tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl} dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethyl-penta-decan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy) propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)pro-pan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl1-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethyl-propan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octa-deca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-die-n-1-yloxylpropan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-methyl}cyclopropyl]octyl} oxy)propan-2-amine,N,N-dimethyl-1-{[-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and(11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or acid or stereoisomer thereof.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in Intl. Publ. No. WO2012/170889, which is herein incorporatedby reference in its entirety

The cationic lipid can routinely be synthesized using methods known inthe art and/or as described in Intl. Publ. Nos. WO2012/040184,WO2011/153120, WO2011/149733, WO2011/090965, WO2011/1043913,WO2011/022460, WO2012/061259, WO 2012/054365, WO2012/044638,WO2010/080724 and WO2010/21865; each of which is herein incorporated byreference in its entirety.

Lipid derivatives can include, for example, at least, the bonding(preferably covalent bonding) of one or more steric stabilizers and/orfunctional groups to the liposomal component after which the stericstabilizers and/or functional groups should be considered part of theliposomal components. Functional groups comprises groups that can beused to attach a liposomal component to another moiety such as aprotein. Such functional groups include, at least, maleimide. Thesesteric stabilizers include at least one from the group consisting ofpolyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside(GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA);poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilicpoly-N-vinylpyrrolidones; L-amino-acid-based polymer; and polyvinylalcohol.

In some embodiments, the provided liposome compositions are formulatedin a lipid-polycation complex. The formation of the lipid-polycationcomplex may be accomplished using methods known in the art and/or asdescribed in U.S. Pub. No. 20120178702, herein incorporated by referencein its entirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine and the cationic peptidesdescribed in International Pub. No. WO2012/013326; herein incorporatedby reference in its entirety. In another embodiment, the providedliposome compositions are formulated in a lipid-polycation complex whichfurther includes a neutral lipid such as, but not limited to,cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

Since the components of a liposome can include any molecule(s)(i.e.,chemical/reagent/protein) that is bound to it, in some embodiments, thecomponents of the provided liposomes include, at least, a memberselected from the group DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG;HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide.In some embodiments, the components of the provided liposomes includeDSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide;cholesterol; cholesterol-PEG; and cholesterol-maleimide. In a preferredembodiment, the liposomal components that make up the liposome comprisesDSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.

In additional embodiments, the liposomes of the liposome compositionsprovided herein comprise oxidized phospholipids. In some embodiments,the liposomes comprise an oxidize phospholipid of a member selected fromthe group consisting of phosphatidylserines, phosphatidylinositols,phosphatidylethanolamines, phosphatidylcholines and1-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments,the phospholipids have unsaturated bonds. In some embodiments, thephospholipids are arachidonic acid containing phospholipids. Inadditional embodiments, the phospholipids are sn-2-oxygenated. Inadditional embodiments, the phospholipids are not fragmented.

In some embodiments, the liposomes of the provided liposome compositionscomprise oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). Theterm “oxPAPC”, as used herein, refers to lipids generated by theoxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine(PAPC), which results in a mixture of oxidized phospholipids containingeither fragmented or full length oxygenated sn-2 residues.Well-characterized oxidatively fragmented species contain a five-carbonsn-2 residue bearing omega-aldehyde or omega-carboxyl groups. Oxidationof arachidonic acid residue also produces phospholipids containingesterified isoprostanes. oxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC andKOOA-PC species, among other oxidized products present in oxPAPC. Infurther embodiments, the oxPAPCs are epoxyisoprostane-containingphospholipids. In further embodiments, the oxPAPC is1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine(5,6-PEIPC),1-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine(PECPC) and/or 1-palmitoyl-2-(epoxy-isoprostaneE2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, thephospholipids have unsaturated bonds. In some embodiments, thephospholipids are arachidonic acid containing phospholipids. Inadditional embodiments, the phospholipids are sn-2-oxygenated. Inadditional embodiments, the phospholipids are not fragmented.

In some embodiments, the liposomes of the provided liposome compositionare pegylated. In some embodiments, the liposome composition is watersoluble. That is, the liposome composition is in the form of an aqueoussolution.

In some embodiments, the liposomes of the provided liposome compositionscomprise a lipid selected from:1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC);1-palmitoyl-2-(9′oxo-nonanoyl)-sn-glycero-3-phosphocholine;1-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine. In furtherembodiments, the liposome comprises PGPC.

In some embodiments, the pH of solutions comprising the liposomecomposition is from pH 2 to 8, or any range therein between. In someembodiments, the pH of solutions comprising the liposome composition isfrom pH 5 to 8, or any range therein between. In some embodiments, thepH of solutions comprising the liposome composition is from pH 6 to 7,or any range therein between. In some embodiments, the pH of solutionscomprising the liposome composition is from 6 to 7.5, from 6.5 to 7.5,from 6.7 to 7.5, or from 6.3 to 7.0, or any range therein between.

In some embodiments, at least one component of the liposome lipidbilayer is functionalized (or reactive). As used herein, afunctionalized component is a component that comprises a reactive groupthat can be used to crosslink reagents and moieties to the lipid. If thelipid is functionalized, any liposome that it forms is alsofunctionalized. In some embodiments, the reactive group is one that willreact with a crosslinker (or other moiety) to form crosslinks. Thereactive group in the liposome lipid bilayer can be located anywhere onthe lipid that allows it to contact a crosslinker and be crosslinked toanother moiety (e.g., a steric stabilizer or targeting moiety). In someembodiments, the reactive group is in the head group of the lipid,including for example a phospholipid. In some embodiments, the reactivegroup is a maleimide group. Maleimide groups can be crosslinked to eachother in the presence of dithiol crosslinkers including but not limitedto dithiolthrietol (DTT).

The liposome can also contain functional lipids and modified lipids asmembrane constituents. Representative, non-limiting examples offunctional lipids include lipid derivatives retained in blood (e.g.,glycophorin, ganglioside GM1, ganglioside GM3, glucuronic acidderivatives, glutaminic acid derivatives, polyglycerin phospholipidderivatives, polyethylene glycol derivatives (methoxypolyethylene glycolcondensates, etc.) such as N-[carbonyl-methoxy polyethylene glyco1-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine,N-[carbonyl-methoxy polyethyleneglycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine,N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-snglycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethyleneglycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine(MPEG 2000-distearoyl phosphatidyl ethanolamine), andN-[carbonyl-methoxy polyethyleneglycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, which arecondensates of phosphoethanolamine and methoxy polyethylene glycol),temperature-sensitive lipid derivatives (e.g., dipalmitoylphosphatidylcholine), pH-sensitive lipid derivatives (e.g., dioleoylphosphatidyl ethanolamine), and the like. Liposomes containing lipidderivatives retained in blood are useful for improving the bloodretention of the liposome, because the liposome becomes difficult tocapture in the liver as a foreign impurity. Similarly, liposomescontaining temperature-sensitive lipid derivatives are useful forcausing destruction of liposome at specific temperatures and/or causingchanges in the surface properties of the liposome. Furthermore, bycombining this with an increase in temperature at the target site, it ispossible to destroy the liposome at the target site, and release thetherapeutic agent at the target site. Liposomes containing pH-sensitivelipid derivatives are useful for enhancing membrane fusion of liposomeand endosome when the liposome is incorporated into cells due to theendocytosis to thereby improve transmission of the therapeutic agent tothe cytoplasm.

It is to be understood that the use of other functionalized lipids,other reactive groups, and other crosslinkers beyond those describedabove is further contemplated. In addition to the maleimide groups,other examples of contemplated reactive groups include but are notlimited to other thiol reactive groups, amino groups such as primary andsecondary amines, carboxyl groups, hydroxyl groups, aldehyde groups,alkyne groups, azide groups, carbonyls, halo acetyl (e.g., iodoacetyl)groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydrylgroups, and pyridyl disulfide groups.

Functionalized and non-functionalized lipids are available from a numberof commercial sources including Avanti Polar Lipids (Alabaster, Ala.)and Lipoid LLC (Newark, N.J.).

In some embodiments, the liposomes of the disclosed lipid compositionsfurther comprise an immunostimulatory agent, a detectable marker, orboth disposed on its exterior surface. The immunostimulatory agent ordetectable marker can be ionically bonded or covalently bonded to theexterior of the liposome, including, for example, optionally to a stericstabilizer component of the liposome.

The terms “immunostimulatory agents” “immunostimulants,”“immune-stimulators,” and the like are used interchangeably herein torefer to substances that stimulate the immune system by inducingactivation or increasing activity of any of its components. Theseimmunostimulatory agents can include one or more of a hapten, anadjuvant, a protein immunostimulating agent, a nucleic acidimmunostimulating agent, and a chemical immunostimulating agent. Manyadjuvants contain a substance designed to stimulate immune responses,such as lipid A, Bordetella pertussis or mycobacterium tuberculosisderived proteins. Certain adjuvants are commercially available as, forexample, Freund's Incomplete Adjuvant and Complete Adjuvant (DifcoLaboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophos-phoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, and -12, and other growth factors, can alsobe used as adjuvants. In a preferred embodiment, the immunostimulant isselected from the group consisting of fluorescein, DNP, beta glucan,beta-1,3-glucan, and beta-1,6-glucan.

A detectable marker may for example, include, at least, a radioisotope,a fluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator, an enzyme, a dye, an ink, a magneticcompound, a biocatalyst or a pigment that is detectable by any suitablemeans known in the art, e.g., magnetic resonance imaging (MRI), opticalimaging, fluorescent/luminescent imaging, or nuclear imaging techniques.

In some embodiments, the immunostimulatory agent and/or detectablemarker is attached to the exterior of the liposome by co-incubating theagent with the liposome. For example, the immunostimulatory agent and/ordetectable marker may be associated with the liposomal membrane byhydrophobic interactions or by an ionic bond such as an avidin/biotinbond or a metal chelation bond (e.g., Ni-NTA). Alternatively, theimmunostimulatory agent or detectable marker may be covalently bonded tothe exterior of the liposome such as, for example, by being covalentlybonded to a liposomal component or to the steric stabilizer which is thePEG.

In some embodiments, liposomes in the provided liposome compositionsfurther comprise an agent that increases the uptake of liposomes into acellular compartment of interest including the cytosol.

In some embodiments, liposomes in the provided liposome compositionscomprise a mitochondrial-targeting agent. In some embodiments, theliposomes comprise triphenylphosphonium (TPP). Methods and mechanismsfor surface functionalizing liposomes with TPP are known in the art(e.g., attaching TPP to the lipid anchor via a peg spacer group andmodifying TPP with a stearyl group (stearyl triphenylphosphonium(STPP)). In some embodiments, the liposomes comprise high-densityocta-arginine. In some embodiments, the liposomes comprise sphingomyelinand/or a sphingomyelin metabolite. Sphingomyelin metabolite used toformulate the liposomes of the present invention can include, forexample ceramide, sphingosine or sphingosine 1-phosphate. In someembodiments, the liposomes comprise Rhodamine 123. In some embodiments,the liposomes comprise, a mitochondria penetrating peptide. In someembodiments, the liposomes comprise, a mitochondria penetrating agentselected from the group: a mitofusin peptide, a mitochondrial targetingsignal peptide, Antennapedia helix III homeodomain cell-penetratingpeptide (ANT) (e.g., comprising RQIKIWFQNRRMKWKKRKKRRQR RR (SEQ IDNO:1), RKKRRXR RRGC where X is any natural or non-natural amino acid(SEQ ID NO:2), CCGCCAAGAAGCG (SEQ ID NO:3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGG CGTGGCGCGGGGGCGTCAT (SEQ ID NO:4),ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO:5), orACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCC CTCCGGGGCGAGCTG (SEQ IDNO:6)), or a mitochondrial penetrating fragment thereof.

In some embodiments, liposomes in the provided liposome compositionscomprise a mitochondria penetrating agent selected from the group: aguanidine-rich peptoid, tetraguanidinium, triguanidinium, diguanidinium,monoguanidinium, a guanidine-rich polycarbamate, a beta-oligoarginine, aproline-rich dendrimer, and a phosphonium salt (e.g.,methyltriphenyl-phosphonium and/or tetraphenylphosphonium).

In some embodiments, liposomes in the provided liposome compositionscomprise sphingomyelin and/or stearyl-octa-arginine. In someembodiments, the liposomes comprise sphingomyelin and/orstearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE,sphingomyelin, stearyl-octa-arginine sphingomyelin andstearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE,sphingomyelin, stearyl-octa-arginine sphingomyelin andstearyl-octa-arginine at a molar ratio of 9:2:1. In some embodiments,the liposomes comprise the MITO-porter system or a variant thereof.

In some embodiments, liposomes in the provided liposome compositionscomprise an agent such as a cell penetrating agent that that facilitatesdelivery of the liposome across a cell membrane and provides theliposome with the ability to bypass the endocytic pathway and the harshenvironment of lysosomes. Cell penetrating agents are known in the artand can routinely be used and adapted for manufacture and use of theprovided liposome compositions. In some embodiments, the cellpenetrating/lysosome bypassing agent is chloroquine. In someembodiments, the cell penetrating agent is a cell penetrating peptide.In some embodiments, liposomes in the provided liposome compositionscomprise a cell penetrating agent selected from the group: RKKRRQRRR(SEQ ID NO:7), GRKKRRQRRRTPQ (SEQ ID NO:8), YGRKKRRQRRR (SEQ ID NO:9),AAVALLPAVLLALLA (SEQ ID NO:10), MGLGLHLLVLAAALQ (SEQ ID NO:11),GALFLGFLGAAGSTM (SEQ ID NO:12), AGYLLGKINLKALAALAKKIL (SEQ ID NO:13),RVIRVWFQNKRCKDKK (SEQ ID NO:14), RQIKIWFQNRRMKWKK (SEQ ID NO:15),GLFEAIAGFIENGWEGMIDG (SEQ ID NO:16), GWTLNSAGYLLGKIN (SEQ ID NO:17),RSQSRSRYYRQRQRS (SEQ ID NO:18), LAIPEQEY (SEQ ID NO:19), LGIAEQEY (SEQID NO:20), LGIPAQEY (SEQ ID NO:21), LGIPEAEY (SEQ ID NO:22), LGIPEQAY(SEQ ID NO:23), LGIAEAEY (SEQ ID NO:24), LGIPEAAY (SEQ ID NO:25),LGIAEQAY (SEQ ID NO:26), LGIAEAAY (SEQ ID NO:27), LLIILRRRIR KQAHAHSK(SEQ ID NO:28), LKALAALAKKIL (SEQ ID NO:29), KLALKLALKALKAALKLA (SEQ IDNO:30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO:31), DHQLNPAF (SEQ ID NO:32),DPKGDPKG (SEQ ID NO:33), VTVTVTVTVTGKGDPKPD (SEQ ID NO:34),RQIKIWFQNRRMKWKK (SEQ ID NO:35), GRKKRRQRRRPPQ (SEQ ID NO:36),GWTLNSAGYLLGKINLKALAAL AKKIL (SEQ ID NO:37), GRKKRRQRRR (SEQ ID NO:38),RRRRRRR (SEQ ID NO:39), RRRRRRRR (SEQ ID NO:40), RRRRRRRRR (SEQ IDNO:41), RRRRRR RRRR (SEQ ID NO:42), RRRRRRRRRRR (SEQ ID NO:43), andYTIWMPENPRPGT PCDIFTNSRGKRASNGGG G(R)n wherein n=2-15 R in the L- and/orD-form (SEQ ID NO:44), or a cell permeating fragment thereof.

In some embodiments, the internal phase of liposomes in the providedliposome compositions comprise platinum-cyclodextrin complexes andfurther comprise a pharmaceutically acceptable carrier such astrehalose. In an additional embodiment, the trehalose is present atabout 5% to 20% weight percent of trehalose or any combination of one ormore lyoprotectants or cryoprotectants at a total concentration of 5% to20%.

In some embodiments, the internal phase of liposomes in the providedliposome compositions comprise platinum-polyglutamate complexes andfurther comprise a pharmaceutically acceptable carrier such astrehalose. In an additional embodiment, the trehalose is present atabout 5% to 20% weight percent of trehalose or any combination of one ormore lyoprotectants or cryoprotectants at a total concentration of 5% to20%. In further non-limiting embodiments, liposomes in the providedliposome compositions enclose an interior space (internal phase). Insome embodiments, the interior space comprises, but is not limited to,an aqueous solution. In some embodiments, the interior space comprises atherapeutic agent-polyglutamate or therapeutic agent-cyclodextrincomplex as provided herein. In additional embodiments, the interiorspace of the liposome comprises a tonicity agent. In some embodiments.In some embodiments, the concentration (weight percent) of the tonicityagent is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range thereinbetween. In some embodiments, the interior space of the liposomeincludes a sugar (e.g., trehalose, maltose, sucrose, lactose, mannose,mannitol, glycerol, dextrose, fructose, etc.). In further embodiments,the concentration (weight percent) of the sugar is 0.1-20%, 1-20%,0.5-15%, 1%-15%, or 1-50%, or any range therein between. In someembodiments, the pH of the interior space of the liposome is from pH 2to 8, or any range therein between. In some embodiments, the pH ofsolutions comprising the liposome composition is from pH 5 to 8, or anyrange therein between. In some embodiments, the pH of solutionscomprising the liposome composition is from pH 6 to 7, or any rangetherein between. In some embodiments, the pH of solutions comprising theliposome composition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5,or from 6.3 to 7.0, or any range therein between. In some embodiments,the interior space comprises buffer. In further embodiments, the buffera buffer selected from HEPES, citrate, or sodium phosphate (e.g.,monobasic and/or dibasic sodium phosphate). In some embodiments, thebuffer is HEPES. In some embodiments, the buffer is citrate. In someembodiments, the buffer is sodium phosphate (e.g., monobasic and/ordibasic sodium phosphate). In some embodiments, the buffer is at aconcentration of 15 to 200 mM, or any range therein between. In yetfurther embodiments, the buffer is at a concentration of between 5 to200 mM, 15-200, between 5 to 100 mM, between 15 to 100 mM, between 5 to50 mM, between 15 to 50 mM, between 5 to 25 mM, between 5 to 20 mM,between 5 to 15 mM, or any range therein between. In some embodiments,the buffer is HEPES at a concentration of 15 to 200 mM, or any rangetherein between. In some embodiments, the buffer is citrate at aconcentration of 15 to 200 mM, or any range therein between. In someembodiments, the buffer is sodium phosphate at a concentration of 15 to200 mM, or any range therein between. In some embodiments, the interiorspace of the liposome comprises a total concentration of sodium acetateand calcium acetate of between 5 mM to 500 mM, or 50 mM to 500 mM, orany range therein between.

In some embodiments, the interior phase of liposomes in the providedliposome compositions comprise liposome includes trehalose. In furtherembodiments, the concentration weight percent of trehalose is 0.1-20%,1-20%, 0.5-15%, 1%-15%, 5-20%, or 1-50%, or any range therein between.In yet further embodiments, the concentration (weight percent) oftrehalose is 1-15%, or any range therein between. In an additionalembodiment, the trehalose is present at about 5% to 20% weight percentof trehalose or any combination of one or more lyoprotectants orcryoprotectants at a total concentration of 5% to 20%.

In some embodiments, the internal phase of liposomes in the providedliposome compositions comprises a buffer. In further embodiments, thebuffer is HEPES buffer or citrate buffer. In some embodiments, thecitrate buffer is at a concentration of between 5 to 200 mM. In someembodiments, the internal phase of the liposome has a pH of between 2.5to 7.5. In some embodiments, the internal phase of the liposome has a pHof between 2.8 to 6. In additional embodiments, the internal phase ofthe liposome comprises sodium acetate and/or calcium acetate. In someembodiments, the internal phase of the liposome comprises a totalconcentration of sodium acetate and calcium acetate of between 5 mM to500 mM, or 50 mM to 500 mM, or any range therein between.

As discussed above, liposomes of the provided liposome composition maycomprise a steric stabilizer. For those embodiments, which incorporate asteric stabilizer, the steric stabilizer may be at least one memberselected from the group consisting of polyethylene glycol (PEG),poly-L-lysine (PLL), monosialoganglioside (GM1), poly(vinyl pyrrolidone)(PVP), poly-(acrylamide) (PAA), poly(2-methyl-2-oxazoline),poly(2-ethyl-2-oxazoline), phosphatidyl polyglycerol,poly[N-(2-hydroxypropyl) methacrylamide], amphiphilicpoly-N-vinylpyrrolidones, L-amino-acid-based polymer, and polyvinylalcohol. In some embodiments, the steric stabilizer or the population ofsteric stabilizer is PEG. In one embodiment, the steric stabilizer is aPEG. In a further embodiment, the PEG has a number average molecularweight (Mn) of 200 to 5000 daltons. These PEG(s) can be of any structuresuch as linear, branched, star or comb structure and are commerciallyavailable.

In some embodiments, the liposome composition comprises liposomes thatfurther contain one or more of an immunostimulatory agent, a detectablemarker and a maleimide disposed on at least one of the PEG and theexterior of the liposome.

In some embodiments, liposomes in the provided liposome compositions aretargeted pegylated liposomes that including an interior space; a (a)therapeutic agent-polyglutamate complex, or (b) therapeuticagent-cyclodextrin complex, disposed within the interior phase (space);and a targeting moiety comprising a protein with specific affinity forat least one folate receptor, and wherein the targeting moiety disposedat the exterior of the liposome. In some embodiments, the medium is anaqueous solution. In some embodiments, the interior space, the exteriorspace (e.g., the medium), or both the interior space and the mediumcontains one or more lyoprotectants or cryoprotectants which are listedabove. In some embodiments, the cryoprotectant is mannitol, trehalose,sorbitol, or sucrose.

In some embodiments, internal phase (interior space) of liposomes in theprovided liposome compositions contains less than 200,000 complexes ofplatinum-based chemotherapeutic agents and one or more polyglutamatemolecules and/or other polyglutamate molecule complexes provided herein.In some embodiments, the internal phase of the liposome contains between10,000 to 100,000 complexes of platinum-based chemotherapeutic agentsand one or more polyglutamate molecule and/or other polyglutamatemolecule complexes provided herein. In further embodiments, the internalphase of the liposome contains between 10,000 to 100,000 complexes ofplatinum-based chemotherapeutic agents and one or more polyglutamatemolecules and/or other polyglutamate molecule complexes provided herein.

In some embodiments, internal phase (interior space) of liposomes in theprovided liposome compositions contains less than 200,000 complexes ofplatinum-based chemotherapeutic agents and cyclodextrin and/or othercyclodextrin complexes provided herein. In some embodiments, theinternal phase of the liposome contains between 10,000 to 100,000complexes of platinum-based chemotherapeutic agents and cyclodextrinand/or other cyclodextrin complexes provided herein. In furtherembodiments, the internal phase of the liposome contains between 10,000to 100,000 complexes of platinum-based chemotherapeutic agents andcyclodextrin and/or other cyclodextrin complexes provided herein.

In some embodiments, liposomes in the liposome composition comprise acomplex formed by one or more polyglutamate molecules and a therapeuticagent. In some embodiments, the therapeutic agent is a cytotoxiccompound or a salt or acid thereof. In a further embodiment, thetherapeutic agent is a chemotherapeutic agent or a salt or acid thereof.In another embodiment, the therapeutic agent is a platinum-basedchemotherapeutic agent. In another embodiment, the therapeutic agent isa taxane-based drug. In further embodiments, the complex is formed byone or more polyglutamate molecules and a therapeutic agent selectedfrom: gemcitabine, a gemcitabine-based therapeutic agent, doxorubicin,an antifolate, an antifolate-based chemotherapeutic, or a salt or acid,acid or free base form thereof. In additional embodiments, the molarratio of the polyglutamate molecule-therapeutic agent in the complex isin the range 1-10:1. In some embodiments, the molar ratio of thepolyglutamate molecule-therapeutic agent in the complex is: 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In some embodiments,the molar ratio of the polyglutamate molecule-therapeutic agent in thecomplex is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or1:>50.

In some embodiments, liposomes in the liposome composition comprise acomplex formed by a cyclodextrin and a therapeutic agent. In someembodiments, the therapeutic agent is a cytotoxic compound or a salt oracid thereof. In a further embodiment, the therapeutic agent is achemotherapeutic agent or a salt or acid thereof. In another embodiment,the therapeutic agent is a platinum-based chemotherapeutic agent. Inanother embodiment, the therapeutic agent is a taxane-based drug. Infurther embodiments, the therapeutic agent of thecyclodextrin/therapeutic agent complex is a member selected from thegroup: gemcitabine, a gemcitabine-based therapeutic agent, doxorubicin,an antifolate, an antifolate-based chemotherapeutic, or a salt or acid,acid or free base form thereof. In additional embodiments, the molarratio of cyclodextrin/therapeutic agent in the complex is in the range1-10:1. In some embodiments, the molar ratio of αPPMX/therapeutic agentin the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10,1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50),or 1:>50. In some embodiments, the molar ratio ofcyclodextrin/therapeutic agent in the complex is: 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, (21-50):1, or >50:1.

In one embodiment the provided liposome compositions comprise liposomesthat are untargeted. That is, the liposome compositions do not havespecific affinity towards a specific epitope expressed on the surface ofa target cell of interest.

In other embodiments, the provided liposome compositions compriseliposomes that are targeted and that comprise a targeting moiety havingan affinity for an epitope (antigen) expressed on the surface of atarget cell of interest. The targeted liposome compositions providefurther improvements to the efficacy and safety of deliveringplatinum-based drugs to hyperproliferative cells such as cancer cells,by specifically delivering platinum payloads to the target cell.

In some embodiments, the internal phase of liposomes in the providedliposome compositions comprise platinum-polyglutamate complexes orplatinum-cyclodextrin complexes, and the external surface of theliposome comprise a targeting moiety comprising a protein with specificaffinity for a surface antigen that is expressed on a target cell ofinterest (e.g., a folate receptor). Such liposomes may generally bereferred to herein as “targeted liposomes,” e.g., liposomes includingone or more targeting moieties or biodistribution modifiers on thesurface of, or otherwise attached to, the liposomes. The targetingmoiety of the targeted liposomes can be any moiety or agent that iscapable of specifically binding a desired target (e.g., an antigentarget expressed on the surface of a cancer cell). In one embodiment,the targeted liposome specifically and preferentially binds to a targeton the surface of a target cell, and whereupon the targeted liposome isinternalized and the cytotoxic payload of the liposome exerts itscytotoxic effect. In further embodiments, the target cell is a cancercell, a tumor cell or a metastatic cell. In some embodiments, thetargeting liposomes are immunoliposomes.

In some embodiments, the disclosure provides a liposome compositioncomprising liposomes encapsulating a complex of a (a) platinum-basedchemotherapeutic agent or a salt thereof and polyglutamate(s) or (b)platinum-based chemotherapeutic agent or a salt thereof and acyclodextrin; and and a targeting moiety attached to one or both of aPEG and the exterior of the liposome, and wherein the targeting moietyhas a specific affinity for a surface antigen on a target cell ofinterest (e.g., a cancer cell). Such liposomes may generally be referredto herein as “targeted liposomes,” e.g., liposomes including one or moretargeting moieties or biodistribution modifiers on the surface of, orotherwise attached to, the liposomes. The targeting moiety of thetargeted liposomes can be any moiety or agent that is capable ofspecifically binding a desired epitope or target (e.g., an antigentarget expressed on the surface of a target cell of interest). In oneembodiment, the targeted liposome specifically and preferentially bindsto a target on the surface of a target cell of interest thatinternalizes the targeted liposome into the cell. In particularembodiments, the target cell is a cancer cell, a tumor cell or ametastatic cell. In some embodiments, the targeted liposome ispegylated.

The term “attach” or “attached” refers, for example, to any type ofbonding such as covalent bonding, ionic bonding (e.g., avidin-biotin)bonding by hydrophobic interactions, and bonding via functional groupssuch as maleimide, or linkers such as PEG. For example, a detectablemarker, a steric stabilizer, a liposome, a liposomal component, animmunostimulating agent may be attached to each other directly, by amaleimide functional group, or by a PEG-malemide group

The composition and origination of the targeting moiety is non-limitingto the scope of this disclosure. In some embodiments, the targetingmoiety attached to the liposome is a polypeptide or peptidomimeticligand. Peptide and peptidomimetic targeting moieties include thosehaving naturally occurring or modified peptides, e.g., D or L peptides;alpha, beta, or gamma peptides; N-methyl peptides; azapeptides; peptideshaving one or more amide, i.e., peptide, linkages replaced with one ormore urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclicpeptides. A peptidomimetic is a molecule capable of folding into adefined three-dimensional structure similar to a natural peptide. Insome embodiments, the peptide or peptidomimetic targeting moiety is 2-50amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50amino acids long

In some embodiments, the targeting moiety polypeptide is at least 40amino acid residues in length. In other embodiments, the targetingmoiety polypeptide is at least 50, 60, 75, 100, 125, 150, 175, 200, 250,or 300 amino acid residues in length.

In additional embodiments, the targeting moiety polypeptide such as anantibody or an antigen-binding antibody fragment that binds a targetantigen with an equilibrium dissociation constant (Kd) in a range of0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE® analysis.

In additional embodiments, the targeting moiety is an antibody or aderivative of the antigen binding domain of an antibody that hasspecific affinity for an epitope on a tumor cell surface antigen ofinterest expressed on the surface of a target cell.

In further embodiments, the targeting moiety is an antibody or afragment of an antibody. In additional embodiments, the targeting moietycomprises one or more of an antibody, a humanized antibody, an antigenbinding fragment of an antibody, a single chain antibody, asingle-domain antibody, a bi-specific antibody, a synthetic antibody, apegylated antibody, and a multimeric antibody. In additionalembodiments, the targeting moiety has the specific affinity for anepitope on a tumor cell surface antigen that is present on a tumor cellbut absent or inaccessible on a non-tumor cell. In some embodiments, thetargeting moiety further comprises one or more of an immunostimulatoryagent, a detectable marker and a maleimide disposed on at least one ofthe PEG and the exterior of the liposome. In some embodiments, thetargeting moiety of the liposome is anionic or neutral. In otherembodiments, the targeting moiety of the liposome is cationic. In someembodiments, the target moiety liposome compositions have a diameter inthe range of 20 nm to 200 nm. In further embodiments, the liposomes havea diameter in the range of 80 nm to 120 nm.

In some embodiments, the targeting moiety is an antibody or an antibodyderivative. In other embodiments, the binding domain of the targetingmoiety polypeptide is not derived from the antigen binding domain of anantibody. In some embodiments, the targeting moiety is a polypeptidederived from a binding scaffold selected from the group consisting of aDARPin, affilin, and armadillo repeat, D domain (see, e.g., WO2016/164308), Z-domain (Affibody), adnectin, lipocalin, affilin,anticalin, knottin, fynomer, atrimer, kunitz domain (see, e.g., WO2004/063337), CTLA4, or avimer (see, e.g., U.S. Publ. Nos. 2004/0175756,2005/0053973, 2005/0048512, and 2006/0008844).

In some embodiments, the liposomes comprise a polypeptide targetingmoiety such as an antibody or an antibody fragment and the targetingmoiety binds a target antigen with an equilibrium dissociation constant(Kd) in a range of 0.5×10⁻¹° to 10×10⁻⁶ as determined using BIACORE®analysis.

In some embodiments, the disclosure provides a liposome compositionwherein the liposome is pegylated and comprises a targeting moietyattached to one or both of a PEG and/or the exterior of the liposome,and wherein the targeting moiety has a specific affinity for a surfaceantigen on a target cell of interest. In some embodiments, the targetingmoiety is a polypeptide. In further embodiments, the targeting moiety isan antibody or a fragment of an antibody. In additional embodiments, thetargeting moiety comprises one or more of an antibody, a humanizedantibody, an antigen binding fragment of an antibody, a single chainantibody, a single-domain antibody, a bi-specific antibody, a syntheticantibody, a pegylated antibody, and a multimeric antibody. In additionalembodiments, the targeting moiety has the specific affinity for anepitope on a tumor cell surface antigen that is present on a tumor cellbut absent or inaccessible on a non-tumor cell. In some embodiments, thetargeting moiety-liposome further comprises one or more of animmunostimulatory agent, a detectable marker and a maleimide disposed onat least one of the PEG and the exterior of the liposome. In someembodiments, the targeting moiety-liposome is anionic or neutral. Inother embodiments, the targeting moiety-liposome is cationic. Inadditional embodiments, the targeting moiety-liposomes have a diameterin the range of 20 nm to 200 nm. In further embodiments, the liposomeshave a diameter in the range of 80 nm to 120 nm.

In some embodiments, the targeting moiety is an antibody or antigenbinding portion of an antibody that specifically binds a targetexpressed on the surface of a target cell of interest. In someembodiments, the targeting moiety is a full-length antibody. In someembodiments, the targeting moiety is an antigen binding portion of anantibody. In some embodiments, the targeting moiety is an scFv. In otherembodiments, the targeting moiety is a Fab. In some embodiments, thetargeting moiety comprises a binding domain derived from the antigenbinding domain of an antibody (e.g., an scFv, Fab, Fab′, F(ab′)2, an Fvfragment, a disulfide-linked Fv (sdFv), a Fd fragment consisting of VHand CH1 domains, an scFv, a minibody, a BiTE, a Tandab, a diabody((VL-VH)₂ or (VH-VL)₂), a single domain antibody (e.g., an sdAb such asa nanobody (either VL or VH)), and a camelid VHH domain) In someembodiments, the targeting moiety comprises one or more complementaritydetermining regions (CDRs) of antibody origin. Examples of suitableantibody-based targeting moieties for the disclosed targeted liposomesinclude a full-length human antibody, a humanized antibody, a chimericantibody, an antigen binding fragment of an antibody, a single chainantibody, a single-domain antibody, a bi-specific antibody, a syntheticantibody, a pegylated antibody and a multimeric antibody. The antibodyof the provided targeted liposomes can have a combination of the abovecharacteristics. For example, a humanized antibody can be an antigenbinding fragment and can be pegylated and multimerized as well.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, and hamster) that have the desiredspecificity, affinity, and capability (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. Nos. 5,225,539 and5,639,641.

In further embodiments, the targeting moiety has specific affinity foran epitope on a surface antigen of a target cell of interest. In someembodiments, the target cell is a cancer cell. In some embodiments, thetarget cell is a tumor cell. In other embodiments, the target cell is animmune cell.

In some embodiments, the targeting moiety-liposome comprises apolypeptide targeting moiety such as an antibody, an antigen bindingportion of an antibody (e.g., a Fab, scFv and a single domain antibody),an aptamer, an affibody and the targeting moiety has a specific affinityfor a target antigen selected from the group consisting of GONMB, CD56,TACSTD2 (TROP2), CEACAM5, Folate receptor-α, Folate receptor-β, Folatereceptor-δ, Mucin 1, STEAP1, Mesothelin, Nectin 4, ENPP3, Guanylylcyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonicanhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6),CGEN-15027, P-Cadherin, Fibronectin Extra-domain B (ED-B), VEGFR2(CD309), Tenascin, Collagen IV, Periostin, endothelin receptor, HER 2,EGFR, CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98.

In some embodiments, the targeting moiety-liposome comprises apolypeptide targeting moiety such as an antibody or antigen bindingportion of an antibody (e.g., a Fab, scFv and a single domain antibody),an aptamer, or an affibody, and the targeting moiety binds a targetantigen with an equilibrium dissociation constant (Kd) in a range of0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE® analysis. In furtherembodiments, the targeting moiety comprises a polypeptide thatspecifically binds a folate receptor. In some embodiments, the folatereceptor bound by the targeting moiety is one or more folate receptorsselected from the group: folate receptor alpha (FR-α), folate receptorbeta (FR-β), and folate receptor delta (FR-δ).

In some embodiments, the targeting moiety has specific affinity for anepitope expressed on a tumor cell surface antigen. The term “tumor cellsurface antigen” refers to an antigen that is common to a specifichyperproliferative disorder such as cancer. In some embodiments, thetargeting moiety has specific affinity for an epitope of a tumor cellsurface antigen that is a tumor associated antigen (TAA). A TAA is anantigen that is found on both tumor and some normal cells. A TAA may beexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or may be normally present atextremely low levels on normal cells but which are expressed at muchhigher levels on tumor cells. Because of the dynamic nature of tumors,in some instances, tumor cells may express unique antigens at certainstages, and at others also express antigens that are also expressed onnon-tumor cells. Thus, inclusion of a certain marker as a TAA does notpreclude it being considered a tumor specific antigen. In someembodiments, the targeting moiety has specific affinity for an epitopeof a tumor cell surface antigen that is a tumor specific antigen (TSA).A TSA is an antigen that is unique to tumor cells and does not occur onother cells in the body. In some embodiments, the targeting moiety hasspecific affinity for an epitope of a tumor cell surface antigenexpressed on the surface of a cancer including but not limited toprimary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer(e.g., NSCLC or SCLC), liver cancer, non-Hodgkin's lymphoma, Hodgkin'slymphoma, leukemias, multiple myeloma, glioblastoma, neuroblastoma,uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladdercancer, kidney cancer, mesothelioma, and adenocarcinomas such as breastcancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancerand other cancers known in the art. In some embodiments, the targetingmoiety has specific affinity for an epitope of a cell surface antigenexpressed on the surface of a cell in the tumor microenvironment (e.g.,and antigen such as VEGFR and TIE1, or TIE2 expressed on endothelialcells and macrophage, respectively, or an antigen expressed on tumorstromal cells such as cancer-associated fibroblasts (CAFs) tumorinfiltrating T cells and other leukocytes, and myeloid cells includingmast cells, eosinophils, and tumor-associated macrophages (TAM).

In some embodiments, the targeted liposome comprises a targeting moietythat has specific affinity for an epitope of a cancer or tumor cellsurface antigen that is preferentially/differentially expressed on atarget cell such as a cancer cell or tumor cell, compared to normal ornon-tumor cells, that is present on a tumor cell but absent orinaccessible on a non-tumor cell. For example, in some situations, thetumor antigen is on the surface of both normal cells and malignantcancer cells but the tumor epitope is only exposed in a cancer cell. Asa further example, a tumor cell surface antigen may experience aconfirmation change in a cancerous state that causes a cancer cellspecific epitope to be present. A targeting moiety with specificaffinity to an epitope on a targetable tumor cell surface antigenprovided herein or otherwise known in the art is useful and isencompassed by the disclosed compositions and methods. In someembodiments, the tumor cell with the tumor cell surface antigen is acancer cell. Examples of such tumor cell surface antigens include,without limitation folate receptor alpha, folate receptor beta andfolate receptor delta.

In further embodiments, the targeting moiety comprises a polypeptidetargeting moiety such as an antibody or an antigen-binding antibodyfragment and the targeting moiety has binding specificity for a folatereceptor. In some embodiments, the targeting moiety binds a folatereceptor with an equilibrium dissociation constant (Kd) in a range of0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE® analysis. In someembodiments, the folate receptor bound by the targeting moiety is one ormore folate receptors selected from the group: folate receptor alpha(FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ).In a further embodiment, the targeting moiety has specific affinity forat least two antigens selected from the group consisting of folatereceptor alpha, folate receptor beta, and folate receptor delta. Inanother embodiment, the targeting moiety has specific affinity forfolate receptor alpha; folate receptor beta; and folate receptor delta.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen that internalizes the targeting moietyupon binding. Numerous cell surface antigens that internalize bindingpartners such as antibodies upon binding are known in the art and areenvisioned to be binding targets for the targeting moieties expressed ontargeted liposomes of the liposome compositions provided herein.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen selected from the group: GONMB,TACSTD2 (TROP2), CEACAMS, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK.

In some embodiments, the targeting moiety has a specific affinity for acell surface antigen(s) derived, from or determined to be expressed on,a specific subject's cancer (e.g., tumor) such as a neoantigen.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen selected from the group consisting ofmannose-6-phosphate receptor, transferrin receptor, and a cell adhesionmolecule (CAM). In further embodiments, the targeting moiety has aspecific affinity for an epitope of a CAM is selected from the groupconsist of: intercellular adhesion molecule (ICAM), platelet-endothelialcell adhesion molecule (PECAM), activated leukocyte cell adhesionmolecule (ALCAM), B-lymphocyte cell adhesion molecule (BL-CAM), vascularcell adhesion molecule (VCAM), mucosal vascular addressin cell adhesionmolecule (MAdCAM), CD44, LFA-2, LFA-3, and basigin.

A discussed herein, folate receptors (FRs) are distinct from reducedfolate carriers (RFCs) and exploit different pathways for bringingfolates and antifolates into cells. In some embodiments, the targetingmoiety specifically binds a folate receptor. In further embodiments, thetargeting moiety specifically binds a folate receptor selected fromfolate receptor alpha, folate receptor beta and folate receptor delta.Antibodies to folate receptor alpha can routinely be generated usingtechniques known in the art. Moreover, the sequences of numerousanti-folate receptor antibodies are in the public domain and/orcommercially available and are readily obtainable.

Murine antibodies against folate receptor are examples of antibodiesthat can be used as targeting moieties of the disclosed targetedliposome is a murine antibody against folate receptor. The sequence ofthese antibodies are known and are described, for example, in U.S. Pat.Nos. 5,646,253; 8,388,972; 8,871,206; and 9,133,275 and in Intl. Appl.Nos. PCT/US2011/056966 and PCT/US2012/046672. For example, based on thesequences disclosed already in the public domain, the gene for theantibodies can be synthesized and placed into a transient expressionvector and the antibody was produced in HEK-293 transient expressionsystem. The antibody can be a complete antibody, a Fab, or any of thevarious antibody variations discussed herein or otherwise known in theart.

In some embodiments, the targeted liposome contains from 1 to 1,000, ormore than 1,000, targeting moieties on its surface. In some embodiments,the targeted liposome contains from 30 to 1,000, 30 to 500, 30 to 250 or30-200, targeting moieties, or any range therein between. In someembodiments, the targeted liposome contains less than 220 targetingmoieties, less than 200 targeting moieties, or less than 175 targetingmoieties. In some embodiments, the targeting moiety is non-covalentlybonded to the outside of the liposome (e.g., via ionic interaction or aGPI anchor).

In some embodiments, the molecules on the outside of the targetedliposome include a lipid, a targeting moiety, a steric stabilizer (e.g.,a PEG), a maleimide, and a cholesterol. In some embodiments, thetargeting moiety is covalently bound via a maleimide functional group.In some embodiments, the targeting moiety is covalently bound to aliposomal component or a steric stabilizer such as a PEG molecule. Insome embodiments, all the targeting moieties of the liposome are boundto one component of the liposome such as a PEG. In other embodiments,the targeting moieties of the targeted liposome are bound to differentcomponents of the liposome. For example, some targeting moieties may bebound to the lipid components or cholesterol, some targeting moietiesmay be bound to the steric stabilizer (e.g., PEG) and still othertargeting moieties may be bound to a detectable marker or to anothertargeting moiety.

In some embodiments, targeted liposomes of the liposome compositionsprovided herein comprise a targeting moiety that has affinity andspecificity (i.e., specifically binds) for an antigen expressed on thesurface of a cancer cell. In further some embodiments, the targetingmoiety of the targeted liposome has affinity and specificity for one ormore antigens selected from the group consisting of folate receptoralpha, folate receptor beta, and folate receptor delta. In oneembodiment, the targeting moiety has specific affinity (i.e.,specifically binds) an antigen selected from the group consisting offolate receptor alpha, folate receptor beta, and folate receptor delta.In a further embodiment, the targeting moiety has specific affinity forat least two antigens selected from the group consisting of folatereceptor alpha, folate receptor beta, and folate receptor delta. Inanother embodiment, the targeting moiety has specific affinity for threeantigens which are, for example, folate receptor alpha; folate receptorbeta; and folate receptor delta. The targeting moiety may have affinityand specificity to an epitope of the antigen because sometimes atargeting moiety does not bind the complete antigen but just an epitopeof many epitopes in an antigen.

In some embodiments, targeted liposomes of the liposome compositionsprovided herein comprise a targeting moiety that is an antibody or aderivative of the antigen binding domain of an antibody that hasspecific affinity for an epitope on a tumor cell surface antigen that ispresent on a tumor cell but absent or inaccessible on a non-tumor cell.For example, in some situations, the tumor antigen is on the surface ofboth normal cells and malignant cancer cells but the tumor epitope isonly exposed in a cancer cell. As a further example, a tumor antigen mayexperience a confirmation change in cancer causing cancer cell specificepitopes to be present. A targeting moiety with specific affinity toepitopes provided herein are useful and are encompassed by the disclosedcompositions and methods. In some embodiments, the tumor cell with thecancer cell specific epitope(s) is a cancer cell. Examples of such tumorcell surface antigens include, folate receptor alpha, folate receptorbeta and folate receptor delta.

There are no particular limitations on the mixing amount (mole fraction)of the phospholipids and/or phospholipid derivatives that are used whenpreparing the liposome. In one embodiment, 10 to 80% relative to theentire liposome membrane composition can be used. In another embodiment,a range of between 30 to 60% can be used.

In addition, there are no particular limitations on the solvent of theliposome internal phase. Exemplary buffer solutions include, withoutlimitation, as phosphate buffer solution, citrate buffer solution, andphosphate-buffered physiological saline solution, and physiologicalsaline water. In the case where buffer solution is used as solvent, itis preferable that the concentration of buffer agent be 5 to 300 mM, 10to 100 mM, or any range in between. There are also no particularlimitations on the pH of the liposome internal phase. In someembodiments, the liposome internal phase has a pH between 2 and 11, 3and 9, 4 and 7, 4 and 5, 2.5 and 7.5, 2.8 and 6.0 and any range inbetween inclusive.

There are no particular limitations on the solvent of the liposomecomposition in the instances where the liposome composition is a liquidformulation. Representative examples include, without limitation, buffersolutions such as phosphate buffer solution, citrate buffer solution,and phosphate-buffered physiological saline solution, and physiologicalsaline water. There are also no particular limitations on the pH of theliposome external phase of the liposome composition. In someembodiments, such as H is between 2 and 11, 3 and 10, 4 and 9, 7.4, 7.0,or any pH higher than that of the liposome internal phase.

In some embodiments, the liposome composition comprises an excipient. Inadditional embodiments, the excipient comprises more than one differentexcipient. In further embodiments, the excipient comprises a sugar or analcohol. In some embodiments, the pharmaceutically excipient is a sugarselected from the group: a monosaccharide such as, glucose, galactose,mannose, fructose, inositol, ribose, or xylose; a disaccharides such as,lactose, sucrose, cellobiose, trehalose, and maltose; a trisaccharidessuch as, raffinose and melezitose; and a polysaccharide. In someembodiments, the pharmaceutically excipient is a sugar alcohol selectedfrom the group: erythritol, xylitol, sortibol, mannitol and maltitol;polyvalent alcohols such as glycerin, diglycerin, polyglycerin,propylene glycol, polypropylene glycol, ethylene glycol, diethyleneglycol, triethylene glycol, polyethylene glycol, ethylene glycolmonoalkylether, diethylene glycol monoalkylether, and 1,3-butyleneglycol. In further embodiments, the solvent of the liposome compositioncomprises a combination of sugar and alcohol excipients.

For purposes of stable long-term storage of liposomes dispersed insolvent, it is preferable to eliminate the electrolyte in the solvent asmuch as possible. Moreover, from the standpoint of chemical stability ofthe lipids, it is preferable to set the pH of the solvent from acidic tothe vicinity of neutral (pH 3.0 to 8.0), and to remove dissolved oxygenthrough nitrogen bubbling. Representative examples of liquid stabilizersinclude, without limitation, normal saline, isotonic dextrose, isotonicsucrose, Ringer's solution, and Hanks' solution. A buffer substance canbe added to provide pH optimal for storage stability. For example, pHbetween about 6.0 and about 7.5, more preferably pH about 6.5, isoptimal for the stability of liposome lipid bilayer lipids, and providesfor excellent retention of the entrapped entities. Histidine,hydroxyethylpiperazine-ethylsulfonate (HEPES), morpholipoethylsulfonate(MES), succinate, tartrate, and citrate, typically at 2-20 mMconcentration, are exemplary buffer substances. Other suitable carriersinclude, e.g., water, buffered aqueous solution, 0.4% NaCl, 0.3%glycine, and the like.

The tonicity of the liposome composition can be adjusted to thephysiological level of 0.25-0.35 mol/kg with glucose or a more inertcompound such as lactose, sucrose, mannitol, or dextrin. Thesecompositions can routinely be sterilized using conventional, knownsterilization techniques, e.g., by filtration. The resulting aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous medium prior to administration. There are no particularlimitations on the concentration of the sugar contained in the liposomecomposition, but in a state where the liposome is dispersed in a solvent(liquid), for example, it is preferable that the concentration of sugarbe 2 to 20% (W/V), and 5 to 10% (W/V) is more preferable. With respectto the concentration of polyvalent alcohol, 1 to 5% (W/V) is preferable,and 2 to 2.5% (W/V) is more preferable.

Solid formulations of the provided liposome compositions can alsoinclude pharmaceutical excipients. Such components can include, forexample, sugar, such as monosaccharides such as glucose, galactose,mannose, fructose, inositole, ribose, and xylose; disaccharides such aslactose, sucrose, cellobiose, trehalose, and maltose; trisaccharidessuch as raffmose and melezitose; polysaccharides such as cyclodextrin;and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, andmaltitol. More preferable are blends of glucose, lactose, sucrose,trehalose, and sorbitol. Even more preferable are blends of lactose,sucrose, and trehalose. By this refers to, solid formulations can bestably stored over long periods. When frozen, it is preferable thatsolid formulations contain polyvalent alcohols (aqueous solutions) suchas glycerin, diglycerin, polyglycerin, propylene glycol, polypropyleneglycol, ethylene glycol, diethylene glycol, triethylene glycol,polyethylene glycol, ethylene glycol monoalkylether, diethylene glycolmonoalkylether and 1,3-butylene glycol. With respect to polyvalentalcohols (aqueous solutions), glycerin, propylene glycol, andpolyethylene glycol are preferable, and glycerin and propylene glycolare more preferable.

Therapeutic Agents

In some embodiments, the disclosure provides a composition comprising adelivery vehicle, such as a liposome composition that comprises apegylated liposome encapsulating a complex of a therapeutic agent and(a) one or more polyglutamate molecule(s), or (b) a cyclodextrin.

There are no particular limitations on the therapeutic agent that iscomplexed with the polyglutamate molecules or cyclodextrin in theliposome compositions disclosed herein as long as the therapeutic agentis capable of being complexed with the the polyglutamate molecules orcyclodextrin and encapsulated in a liposome in a manner sufficient todeliver an effective amount of the complexed therapeutic agent to atarget cell of interest. For example, it is known that α-cyclodextrinhas an internal phase pore diameter size of 0.45-0.6, β-cyclodextrin hasan internal phase pore diameter size of 0.6 to 0.8 nm, andγ-cyclodextrin has an internal phase pore diameter size of 0.8 to 0.95nm. The cyclodextrin can be chosen to match the size of the therapeuticagent to allow for encapsulation. With respect to the molecular weightof the therapeutic agent, a range of 100 to 2,000 daltons is preferable.

As described above, modifications to the non-carbon cyclodextrin groups(e.g., hydroxyl groups) can be selected to modulate intermolecularinteractions between the cyclodextrin and the therapeutic agent tothereby modulate the stability, bioavailability, toxicity, and efficacyof the complexed therapeutic agent.

In some embodiments, the therapeutic agent is anantiproliferative/antineoplastic drug. In some embodiments, thetherapeutic agent is a member selected from the group: an alkylatingagent, an antimetabolite, an antibiotic, an antitimitotic agent, aproteasome inhibitor, and a topoisomerase inhibitor. In furtherembodiments, the therapeutic agent is a member selected from the groupconsisting of cyclophosphamide, bendamustin, melphalan, chlorambucil,nitrogen mustard, temozolamide, busulphan, a nitrosourea, gemcitabine,an antifolate, a fluoropyrimidine, 5-fluorouracil, tegafur, raltitrexed,methotrexate, pemetrexed, cytosine arabinoside, hydroxyurea, ananthracycline (e.g., adriamycin, bleomycin, doxorubicin, daunomycin,epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin), avinca alkaloid (e.g., vincristine, vinblastine, vindesine andvinorelbine), a taxoid (e.g., taxol, taxotere, and a polokinaseinhibitor); carfilzomib, bortezomib, and a epipodophyllotoxin (e.g.,etoposide, teniposide, amsacrine, topotecan, mitoxantrone andcamptothecin.

Gemcitabine

In particular embodiments, the therapeutic agent is gemcitabine (dFdC)or a gemcitabine-based therapeutic agent. Gemcitabine is a marketedanti-metabolic nucleoside analog prodrug used first line to treatvarious solid tumors including non-small-cell lung cancer and pancreaticcancer. Gemcitabine is hydrophilic and relies on nucleoside transportersto cross cell membranes. After entering the cell, gemcitabine (dFdC) isconverted into gemcitabine monophosphate (dFdCMP) by deoxycytidinekinase (DCK) during a crucial and rate-limiting step. Subsequently, twomore phosphates are added by other enzymes to form pharmacologicallyactive gemcitabine triphosphate (dFdCTP), which competes with thenatural substrates for incorporation into DNA and inhibits nucleotidemetabolism.

The development of resistance and systemic toxicity often occur when theintracellular conversion of gemcitabine to its active phosphorylatedform is not efficient. Unfortunately, phosphorylated gemcitabine-basedagents are generally known to be unstable and unable to permeate cellmembranes. Moreover, as reflected by the gemcitabine label, the systemictoxicities associated with gemcitabine treatment include the suppressionof bone marrow function, the loss of white blood cells, red blood cells,and platelets, and loss of red blood cells, and severe lung conditionslike pulmonary edema, pneumonia, and adult respiratory distresssyndrome, among others.

As used herein, the term “gemcitabine” refers to the stable salts, acidsand free base forms of gemcitabine(2′-Deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzoxy-L-alaninyl)]phosphate(dFdC)) and metabolites of gemcitabine as represented in formula IV.

In one aspect, the disclosure provides compositions comprising a complexcontaining a gemcitabine and (a) cyclodextrin or (b) a cationictherapeutic agent. In some embodiments, the cationic therapeutic agentis a platinum-based drug. In some embodiments, thegemcitabine-platinum-based drug complex comprises cisplatin or a saltthereof. In some embodiments, the platinum-based drug complex comprisesoxaliplatin, or a salt thereof.

As used herein, the term “gemcitabine-based liposome composition” refersa liposome compositions that contains a complex of an gemcitabine-basedchemotherapeutic agent (e.g., dFdC and dFdCTP) and a cyclodextrin,and/or a liposome compositions that contains a complex of angemcitabine-based chemotherapeutic agent and a cyclodextrin and thatalso contains a complex of a platinum-based chemotherapeutic agent and acyclodextrin, unless otherwise indicated. In some embodiments, theplatinum-based drug complex comprises stratoplatin, paraplatin,platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin,triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin,platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK121, CI-973,DWA 2114R, NDDP, or dedaplatin, or a salt thereof. Compositionscomprising delivery vehicles such as liposomes that contain/encapsulatethe therapeutic agent complexes are also provided, as are methods ofmaking and using the provided compositions to treat hyperproliferativediseases such as cancer. In some embodiments, the disclosure providesliposome compositions that comprise a liposome encapsulating a complexof a platinum-based chemotherapeutic agent (e.g., cisplatin andoxaliplatin, or a salt thereof) and one or more gemcitabine molecuels(e.g., dFdC and dFdCTP).

In some embodiments, the gemcitabine-based therapeutic agent isgemcitabine (dFdC)(including stable salt and free base forms ofgemcitabine). In some embodiments, the gemcitabine-based therapeuticagent is a phosphorylated gemcitabine (including stable salt and freebase forms of phosphorylated gemcitabine). In some embodiments, thegemcitabine-based therapeutic agent is gemcitabine monophosphate(dFdCMP)(including stable salt and free base forms thereof). In someembodiments, the gemcitabine-based therapeutic agent is gemcitabinediphosphate (dFdCDP)(including stable salt and free base forms thereof).In some embodiments, the gemcitabine-based therapeutic agent isgemcitabine triphosphate (dFdCTP)(including stable salt and free baseforms thereof). These liposome compositions provide improvements to theefficacy and safety of delivering gemcitabine-based agents to cancercells by providing the preferential delivery of a more cytotoxic payload(e.g., phosphorylated gemcitabine such as dFdCMP, dFdCDP, and dFdCTP)compared to the cytotoxicity of the corresponding gemcitabine-basedagent delivered in its uncomplexed and non-liposome encapsulated state.

In a distinct aspect, the disclosure provides liposome compositions thatcontain liposomes encapsulating a complex of a gemcitabine-basedchemotherapeutic agent and a cyclodextrin such as a derivatized betacyclodextrin. In some embodiments, the gemcitabine-based therapeuticagent is gemcitabine (dFdC)(including stable salt and free base forms ofgemcitabine). In some embodiments, the gemcitabine-based therapeuticagent is a phosphorylated gemcitabine (including stable salt and freebase forms of phosphorylated gemcitabine). In some embodiments, thegemcitabine-based therapeutic agent is gemcitabine monophosphate(dFdCMP)(including stable salt and free base forms thereof). In someembodiments, the gemcitabine-based therapeutic agent is gemcitabinediphosphate (dFdCDP)(including stable salt and free base forms thereof).In some embodiments, the gemcitabine-based therapeutic agent isgemcitabine triphosphate (dFdCTP)(including stable salt and free baseforms thereof). These liposome compositions provide improvements to theefficacy and safety of delivering gemcitabine-based agents to cancercells by providing the preferential delivery of a more cytotoxic payload(e.g., phosphorylated gemcitabine such as dFdCMP, dFdCDP, and dFdCTP)compared to the cytotoxicity of the corresponding gemcitabine-basedagent delivered in its uncomplexed and non-liposome encapsulated state.

Accordingly, in one embodiment, the disclosure provides a liposomecomposition comprising liposomes encapsulating a complex of agemcitabine-based agent and a cyclodextrin. In some embodiments, theliposomes are pegylated. In some embodiments, the gemcitabine-basedagent is gemcitabine (dFdC)(including stable salt and free base forms ofgemcitabine). In some embodiments, the gemcitabine-based agent is aphosphorylated gemcitabine (including stable salt and free base forms ofphosphorylated gemcitabine). In some embodiments, the gemcitabine-basedtherapeutic agent is gemcitabine monophosphate (dFdCMP)(including stablesalt and free base forms thereof). In some embodiments, thegemcitabine-based therapeutic agent is gemcitabine diphosphate(dFdCDP)(including stable salt and free base forms thereof). In someembodiments, the gemcitabine-based therapeutic agent is gemcitabinetriphosphate (dFdCTP)(including stable salt and free base formsthereof).

The cyclodextrin complexed with the gemcitabine-based therapeutic agentin the provided liposome compositions can be derivatized orunderivatized. In some embodiments, the cyclodextrin is derivatized. Infurther embodiments, the cyclodextrin is a derivatized beta-cyclodextrin(e.g., a hydroxypropyl beta-cyclodextrin (HP-beta-CD), and a sulfobutylether beta-CD (SBE)-beta-cyclodextrin). In some embodiments, thecyclodextrin is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more 2-hydroxylpropyl-3- group substitutions ofhydroxy groups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkylether group substitutions of hydroxy groups. In further embodiments, thecyclodextrin is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more sulfobutyl ether group substitutions ofhydroxy groups.

In some embodiments, the cyclodextrin complexed with thegemcitabine-based therapeutic agent is a derivatized cyclodextrin ofFormula I:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branched C₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group.

In some embodiments, the cyclodextrin complexed with thegemcitabine-based therapeutic agent is a derivatized cyclodextrin ofFormula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca²⁺, or Mg⁺², and ammonium ions andamine cations such as the cations of (C₁-C₆)-alkylamines, piperidine,pyrazine, (C₁-C₆)-alkanolamine and (C₄-C₈)-cycloalkanolamine.

In some embodiments, the liposomes in the liposome composition comprisebetween 100 to 100,000 gemcitabine-based therapeutic agent/cyclodextrincomplexes. In additional embodiments, the liposome composition comprisesliposomes that have a diameter in the range of 20 nm to 200 nm. Inadditional embodiments, the liposome composition comprises liposomesthat have a diameter in the range of 20 nm to 200 nm. In someembodiments, liposomes in the composition comprise between 100 to100,000 gemcitabine molecules. In some embodiments, liposomes in thecomposition are anionic or neutral. In further embodiments, liposomes inthe composition have a zeta potential that is less than or equal tozero. In further embodiments, liposomes in the composition has a zetapotential that is between 0 to −150 mV. In other embodiments, liposomesin the composition are cationic. In further embodiments, liposomes inthe composition have a zeta potential that is between 1 and 100 mV.

The liposomes in the provided gemcitabine-based liposome compositionsare preferably pegylated. In some embodiments, the polyethylene glycolof the liposomes has a number average molecular weight (Mn) of 200 to5000 daltons. The internal phase of the liposome has a pH in the rangeof 2.8-6.8. In some embodiments, the internal phase of the liposomecomprises trehalose (e.g., 5% to 20% weight of trehalose).

In additional embodiments, the gemcitabine-based liposome compositioncomprises liposomes that contain a targeting moiety. In someembodiments, the targeting moiety is attached to one or both of a PEGand the exterior of the liposome. In additional embodiments, thetargeting moiety has a specific affinity for a surface antigen on atarget cell of interest. In some embodiments, the targeting moiety is apolypeptide. In additional embodiments, the targeting moiety is anantibody, a humanized antibody, an antigen binding fragment of anantibody, a single chain antibody, a single-domain antibody, abi-specific antibody, a synthetic antibody, a pegylated antibody, or amultimeric antibody. In some embodiments, the liposome compositioncomprises liposomes that contain from 30 to 500 targeting moieties. Infurther embodiments, the liposome composition comprises liposomes thatcontain from 30 to 200 targeting moieties.

In some embodiments, the gemcitabine-based liposome compositioncomprises liposomes that further contain one or more of animmunostimulatory agent, a detectable marker and a maleimide disposed onat least one of the PEG and the exterior of the liposome.

In some embodiments, the disclosed gemcitabine-based liposomecompositions further comprise a second complex formed by a therapeuticagent or a salt thereof, and a cyclodextrin. In some embodiments, thetherapeutic agent of the second complex is a member selected from thegroup: gemcitabine, and doxorubicin (including salts and free formsthereof). The cyclodextrin of the second complex in thegemcitabine-based liposome compositions can be derivatized orunderivatized. In some embodiments, the cyclodextrin is derivatized. Infurther embodiments, the cyclodextrin is a derivatized beta-cyclodextrin(e.g., a hydroxypropyl beta-cyclodextrin (HP-beta-CD), and a sulfobutylether beta-CD (SBE)-beta-cyclodextrin). In some embodiments, thecyclodextrin of the second complex in the gemcitabine-based liposomecomposition is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more 2-hydroxylpropyl-3- group substitutions ofhydroxy groups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkylether group substitutions of hydroxy groups. In further embodiments, thecyclodextrin of the second complex is a derivatized beta-cyclodextrincomprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfobutyl ethergroup substitutions of hydroxy groups.

In some embodiments, the cyclodextrin of the second complex contained inthe liposomes of the gemcitabine-based liposome composition (i.e., thecomplex formed by a therapeutic agent (including a salt and free formthereof), and a cyclodextrin) is a derivatized cyclodextrin of FormulaI:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branched C₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group.

In some embodiments, the cyclodextrin of the second complex contained inthe liposomes of the gemcitabine-based liposome composition is aderivatized cyclodextrin of Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺², and ammonium ions andamine cations such as the cations of (C₁-C₆)-alkylamines, piperidine,pyrazine, (C₁-C₆)-alkanolamine and (C₄-C₈)-cycloalkanolamine.

In some embodiments, the liposomes in the gemcitabine-based liposomecomposition comprise between 100 to 100,000 of the second complex formedby the therapeutic agent, and the cyclodextrin. In additionalembodiments, the liposome composition comprises liposomes that have adiameter in the range of 20 nm to 200 nm. In some embodiments, liposomesin the composition comprise between 100 to 100,000 gemcitabine-basedagent/cyclodextrin complexes. In some embodiments, the cyclodextrin ofthe second complex is different from the cyclodextrin of thegemcitabine-based agent complex. In some embodiments, the liposomecomprises the same cyclodextrin in the second complex and thegemcitabine agent-based complex. In some embodiments, the liposomecomprises a cyclodextrin in the second complex that is different fromthe cyclodextrin of the gemcitabine-based agent complex.

According to some embodiments, the liposomes of the gemcitabine-basedliposome compositions comprise oxidized phospholipids. In furtherembodiments, the phospholipids that are oxidized are a member selectedfrom the group consisting of phosphatidylserines, phosphatidylinositols,phosphatidylethanolamines, phosphatidylcholines and1-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate.

In additional embodiments, the gemcitabine-based liposome compositionscomprise oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylchloine (OxPAPC). Infurther embodiments, the oxPAPCs are epoxyisoprostane-containingphospholipids. In further embodiments, the oxPAPC is1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine(5,6-PEIPC),1-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine(PECPC) and/or 1-palmitoyl-2-(epoxy-isoprostaneE2)-sn-glycero-4-phosphocholine (PEIPC).

According to some embodiments, the provided gemcitabine-based liposomecompositions further comprise one or more of an immunostimulatory agent,a detectable marker and a maleimide, wherein the immunostimulatoryagent, the detectable marker or the maleimide is attached to the PEG orthe exterior of the liposome. In some embodiments, the immunostimulatingagent is at least one a member selected from the group: a proteinimmunostimulating agent; a nucleic acid immunostimulating agent; achemical immunostimulating agent; a hapten; and an adjuvant. In someembodiments, the immunostimulating agent is at least one selected fromthe group: a fluorescein; a fluorescein isothiocyanate (FITC); a DNP; abeta glucan; a beta-1,3-glucan; and a beta-1,6-glucan. In someembodiments, the immunostimulatory agent and the detectable marker isthe same. In some embodiments, the liposome composition comprises ahapten. In further embodiments, the hapten comprises one or more offluorescein or Beta 1,6-glucan.

In some embodiments, the liposomes of the disclosed gemcitabine-basedliposome compositions further comprises at least one cryoprotectantselected from the group consisting of mannitol; trehalose; sorbitol; andsucrose. In additional embodiments, the provided liposomal compositionis in unit dosage form. In some embodiments, pharmaceutical compositionscomprising the liposome compositions disclosed herein are provided.

In some embodiments, the disclosure is directed to the use of thedisclosed gemcitabine-based liposome compositions in the treatment ofdisease. In some embodiments, the disclosure is directed to use of theliposome compositions in the manufacture of a medicament for thetreatment of disease.

In some embodiments, the disclosure provides a method of killing ahyperproliferative cell comprising contacting a hyperproliferative cellwith a disclosed gemcitabine-based liposome composition. In furtherembodiments, the hyperproliferative cell is a cancer cell.

In some embodiments, the disclosure provides a method for treating orpreventing disease in a subject needing such treatment or prevention,the method comprising administering an effective amount of agemcitabine-based liposome composition provided herein to a subject inneed thereof. In some embodiments, the administration is parenteral. Insome embodiments, the administration is intravenous. In someembodiments, the administration is subcutaneous

In additional embodiments, the disclosure provides a method for treatingcancer in a subject, comprising administering an effective amount of agemcitabine-based liposome composition disclosed herein to a subjecthaving or at risk of having cancer. In further embodiments, the canceris a member selected from the group: lung cancer, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, head and neck cancer,gastric cancer, gastrointestinal cancer, colorectal cancer, esophagealcancer, cervical cancer, liver cancer, kidney cancer, biliary ductcancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldyscrasias. In some embodiments, the administration is parenteral. Insome embodiments, the administration is intravenous. In someembodiments, the administration is subcutaneous.

In other embodiments, the disclosure provides a method for treatingcancer in a subject, comprising administering an effective amount of agemcitabine-based liposome composition to a tumor expressing an antigenon its surface, the method comprising: administering a gemcitabine-basedliposome composition disclosed herein to a subject having a tumorexpressing the antigen in an amount to deliver a therapeuticallyeffective dose of the liposome composition to the tumor. In someembodiments, the administration is parenteral. In some embodiments, theadministration is intravenous. In some embodiments, the administrationis subcutaneous.

A method for treating cancer that comprises administering an effectiveamount of a gemcitabine-based liposome composition disclosed herein to asubject, wherein the liposome comprises a targeting moiety with specificaffinity for an antigen expressed on the surface of a cancer cell, andwherein the subject has or is at risk of having a cancer cell thatexpresses the antigen. In further embodiments, the antigen is a folatereceptor. In some embodiments, the administration is parenteral. In someembodiments, the administration is intravenous. In some embodiments, theadministration is subcutaneous.

Also provided is maintenance therapy that comprises administering aneffective amount of a gemcitabine-based liposome composition disclosedherein to a subject that is undergoing or has undergone cancer therapy.In some embodiments, the administration is parenteral. In someembodiments, the administration is intravenous. In some embodiments, theadministration is subcutaneous.

Doxorubicin

In additional particular embodiments, the therapeutic agent isdoxorubicin (DOX). Doxorubicin is an anthracyline drug routinely used inthe treatment of several cancers including breast, lung, gastric,bladder, ovarian, thyroid, non-Hodgkin's and Hodgkin's lymphoma, acutelymphocytic leukemia, multiple myeloma, sarcoma (e.g., Kaposi'ssarcoma), and pediatric cancers. However, its clinical application islimited due to severe side effects and the accompanying drug resistance.In particular, a major limitation for the use of doxorubicin iscardiotoxicity, with the total cumulative dose being the only criteriacurrently used to predict the toxicity. Doxorubicin is often usedtogether with other chemotherapy agents. Commonly useddoxorubicin-containing regimens include AC (adriamycin,cyclophosphamide), TAC (taxotere, AC), ABVD (Adriamycin, bleomycin,vinblastine, dacarbazine), BEACOPP, CHOP (cyclophosphamide,hydroxydaunorubicin, vincristine, prednisone) and FAC (5-fluorouracil,adriamycin, cyclophosphamide).

A pegylated (polyethylene glycol coated) liposome-encapsulated form ofdoxorubicin, is sold as DOXIL®. This product was developed to treatKaposi's sarcoma, an AIDS-related cancer that causes the growth oflesions under the skin, and in the lining of the mouth, nose and throat.However, leakage of the doxorubicin in Doxil has been reported to beassociated with uncomfortable and painful adverse events that includeredness, tenderness, and peeling of the skin.

As used herein, the term “doxorubicin” refers to the stable salts, acidsand free base forms of doxorubicin (e.g., doxorubicin HCl). In someembodiments, the disclosure the disclosure provides compositionscomprising a complex containing a doxorubicin and (a) one or morepolyglutamate molecules, or (b) cyclodextrin.

As used herein, the term “doxorubicin-based liposome composition” refersa liposome compositions that contains a complex of an antifolate-basedchemotherapeutic agent and a cyclodextrin, and/or a liposomecompositions that contains a complex of an antifolate-basedchemotherapeutic agent and a cyclodextrin and that also contains acomplex of a platinum-based chemotherapeutic agent and a cyclodextrin,unless otherwise indicated.

In one embodiment, the disclosure provides a liposome compositioncomprising liposomes encapsulating a complex of doxorubicin (includingstable salt and free base forms of doxorubicin, such as doxorubicin HCl)and (a) one or more polyglutamates or (b) a cyclodextrin. In someembodiments, the liposome is pegylated.

In some embodiment, the disclosure provides a liposome compositioncomprising liposomes encapsulating a complex of doxorubicin and one ormore polyglutamates (e.g., as described herein).

In another embodiment, the disclosure provides a liposome compositioncomprising liposomes encapsulating a complex of doxorubicin and acyclodextrin. The cyclodextrin complexed with doxorubicin in theprovided liposome compositions can be derivatized or underivatized. Insome embodiments, the cyclodextrin is derivatized. In furtherembodiments, the cyclodextrin is a derivatized beta-cyclodextrin (e.g.,a hydroxypropyl beta-cyclodextrin (HP-beta-CD), and a sulfobutyl etherbeta-CD (SBE)-beta-cyclodextrin). In some embodiments, the cyclodextrinis a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more 2-hydroxylpropyl-3- group substitutions of hydroxygroups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkyl ether groupsubstitutions of hydroxy groups. In further embodiments, thecyclodextrin is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more sulfobutyl ether group substitutions ofhydroxy groups.

In some embodiments, the cyclodextrin complexed with doxorubicin is aderivatized cyclodextrin of Formula I:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branched C₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group.

In some embodiments, the cyclodextrin complexed with doxorubicin is aderivatized cyclodextrin of Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺², and ammonium ions andamine cations such as the cations of (C1-C6)-alkylamines, piperidine,pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine.

In some embodiments, the liposomes in the liposome composition comprisebetween 100 to 100,000 doxorubicin/cyclodextrin complexes. In additionalembodiments, the liposome composition comprises liposomes that have adiameter in the range of 20 nm to 200 nm. In some embodiments, liposomesin the composition comprise between 100 to 100,000 doxorubicinmolecules. In some embodiments, liposomes in the composition are anionicor neutral. In further embodiments, liposomes in the composition have azeta potential that is less than or equal to zero. In furtherembodiments, liposomes in the composition has a zeta potential that isbetween 0 to −150 mV. In other embodiments, liposomes in the compositionare cationic. In further embodiments, liposomes in the composition havea zeta potential that is between 1 and 100 mV.

The liposomes in the provided doxorubicin-based liposome compositionsare preferably pegylated. In some embodiments, the polyethylene glycolof the liposomes has a number average molecular weight (Mn) of 200 to5000 daltons. The internal phase of the liposome has a pH in the rangeof 2.8-6.8. In some embodiments, the internal phase of the liposomecomprises trehalose (e.g., 5% to 20% weight of trehalose).

In additional embodiments, the doxorubicin-based liposome compositioncomprises liposomes that contain a targeting moiety. In someembodiments, the targeting moiety is attached to one or both of a PEGand the exterior of the liposome. In additional embodiments, thetargeting moiety has a specific affinity for a surface antigen on atarget cell of interest. In some embodiments, the targeting moiety is apolypeptide. In additional embodiments, the targeting moiety is anantibody, a humanized antibody, an antigen binding fragment of anantibody, a single chain antibody, a single-domain antibody, abi-specific antibody, a synthetic antibody, a pegylated antibody, or amultimeric antibody. In some embodiments, the liposome compositioncomprises liposomes that contain from 30 to 500 targeting moieties. Infurther embodiments, the liposome composition comprises liposomes thatcontain from 30 to 200 targeting moieties.

In some embodiments, the doxorubicin-based liposome compositioncomprises liposomes that further contain one or more of animmunostimulatory agent, a detectable marker and a maleimide disposed onat least one of the PEG and the exterior of the liposome.

In some embodiments, the disclosed doxorubicin-based liposomecompositions further comprise a second complex formed by a therapeuticagent or a salt thereof, and a cyclodextrin. In some embodiments, thetherapeutic agent of the second complex is a member selected from thegroup: gemcitabine, and an antifolate (including salts and free formsthereof). The cyclodextrin of the second complex in thedoxorubicin-based liposome compositions can be derivatized orunderivatized. In some embodiments, the cyclodextrin is derivatized. Infurther embodiments, the cyclodextrin is a derivatized beta-cyclodextrin(e.g., a hydroxypropyl beta-cyclodextrin (HP-beta-CD), and a sulfobutylether beta-CD (SBE)-beta-cyclodextrin). In some embodiments, thecyclodextrin of the second complex in the doxorubicin-based liposomecomposition is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more 2-hydroxylpropyl-3- group substitutions ofhydroxy groups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkylether group substitutions of hydroxy groups. In further embodiments, thecyclodextrin of the second complex is a derivatized beta-cyclodextrincomprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfobutyl ethergroup substitutions of hydroxy groups.

In some embodiments, the cyclodextrin of the second complex contained inthe liposomes of the doxorubicin-based liposome composition (i.e., thecomplex formed by a therapeutic agent (including a salt and free formthereof), and a cyclodextrin) is a derivatized cyclodextrin of FormulaI:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branched C₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group.

In some embodiments, the cyclodextrin of the second complex contained inthe liposomes of the doxorubicin-based liposome composition is aderivatized cyclodextrin of Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺², and ammonium ions andamine cations such as the cations of (C1-C6)-alkylamines, piperidine,pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine.

In some embodiments, the liposomes in the doxorubicin-based liposomecomposition comprise between 100 to 100,000 of the second complex formedby the therapeutic agent, and the cyclodextrin. In additionalembodiments, the liposome composition comprises liposomes that have adiameter in the range of 20 nm to 200 nm. In some embodiments, liposomesin the composition comprise between 100 to 100,000doxorubicin/cyclodextrin complexes. In some embodiments, thecyclodextrin of the second complex is different from the cyclodextrin ofthe doxorubicin/cyclodextrin complex. In some embodiments, the liposomecomprises the same cyclodextrin in the second complex and doxorubicincomplex. In some embodiments, the liposome comprises a cyclodextrin inthe second complex that is different from the cyclodextrin of thedoxorubicin complex.

According to some embodiments, the liposomes of the doxorubicin-basedliposome compositions comprise oxidized phospholipids. In furtherembodiments, the phospholipids that are oxidized are a member selectedfrom the group consisting of phosphatidylserines, phosphatidylinositols,phosphatidylethanolamines, phosphatidylcholines and1-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate.

In additional embodiments, the doxorubicin-based liposome compositionscomprise oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylchloine (OxPAPC). Infurther embodiments, the oxPAPCs are epoxyisoprostane-containingphospholipids. In further embodiments, the oxPAPC is1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine(5,6-PEIPC),1-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine(PECPC) and/or 1-palmitoyl-2-(epoxy-isoprostaneE2)-sn-glycero-4-phosphocholine (PEIPC).

According to some embodiments, the provided doxorubicin-based liposomecompositions further comprise one or more of an immunostimulatory agent,a detectable marker and a maleimide, wherein the immunostimulatoryagent, the detectable marker or the maleimide is attached to the PEG orthe exterior of the liposome. In some embodiments, the immunostimulatingagent is at least one is a member selected from the group: a proteinimmunostimulating agent; a nucleic acid immunostimulating agent; achemical immunostimulating agent; a hapten; and an adjuvant. In someembodiments, the immunostimulating agent is at least one selected fromthe group: a fluorescein; a fluorescein isothiocyanate (FITC); a DNP; abeta glucan; a beta-1,3-glucan; and a beta-1,6-glucan. In someembodiments, the immunostimulatory agent and the detectable marker isthe same. In some embodiments, the liposome composition comprises ahapten. In further embodiments, the hapten comprises one or more offluorescein or Beta 1,6-glucan.

In some embodiments, the liposomes of the disclosed doxorubicin-basedliposome compositions further comprises at least one cryoprotectantselected from the group consisting of mannitol; trehalose; sorbitol; andsucrose. In additional embodiments, the provided liposomal compositionis in unit dosage form. In some embodiments, pharmaceutical compositionscomprising the liposome compositions disclosed herein are provided.

In some embodiments, the disclosure is directed to the use of thedisclosed doxorubicin-based liposome compositions in the treatment ofdisease. In some embodiments, the disclosure is directed to use of theliposome compositions in the manufacture of a medicament for thetreatment of disease.

In some embodiments, the disclosure provides a method of killing ahyperproliferative cell comprising contacting a hyperproliferative cellwith a disclosed doxorubicin-based liposome composition. In furtherembodiments, the hyperproliferative cell is a cancer cell.

In some embodiments, the disclosure provides a method for treating orpreventing disease in a subject needing such treatment or prevention,the method comprising administering an effective amount of adoxorubicin-based liposome composition provided herein to a subject inneed thereof. In some embodiments, the administration is parenteral. Insome embodiments, the administration is intravenous. In someembodiments, the administration is subcutaneous

In additional embodiments, the disclosure provides a method for treatingcancer in a subject, comprising administering an effective amount of adoxorubicin-based liposome composition disclosed herein to a subjecthaving or at risk of having cancer. In further embodiments, the canceris a member selected from the group: lung cancer, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, head and neck cancer,gastric cancer, gastrointestinal cancer, colorectal cancer, esophagealcancer, cervical cancer, liver cancer, kidney cancer, biliary ductcancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldyscrasias. In some embodiments, the administration is parenteral. Insome embodiments, the administration is intravenous. In someembodiments, the administration is subcutaneous.

In other embodiments, the disclosure provides a method for treatingcancer in a subject, comprising administering an effective amount of adoxorubicin-based liposome composition to a tumor expressing an antigenon its surface, the method comprising: administering a doxorubicin-basedliposome composition disclosed herein to a subject having a tumorexpressing the antigen in an amount to deliver a therapeuticallyeffective dose of the liposome composition to the tumor. In someembodiments, the administration is parenteral. In some embodiments, theadministration is intravenous. In some embodiments, the administrationis subcutaneous.

A method for treating cancer that comprises administering an effectiveamount of a doxorubicin-based liposome composition disclosed herein to asubject, wherein the liposome comprises a targeting moiety with specificaffinity for an antigen expressed on the surface of a cancer cell, andwherein the subject has or is at risk of having a cancer cell thatexpresses the antigen. In further embodiments, the antigen is a folatereceptor. In some embodiments, the administration is parenteral. In someembodiments, the administration is intravenous. In some embodiments, theadministration is subcutaneous

Also provided is maintenance therapy that comprises administering aneffective amount of a doxorubicin-based liposome composition disclosedherein to a subject that is undergoing or has undergone cancer therapy.In some embodiments, the administration is parenteral. In someembodiments, the administration is intravenous. In some embodiments, theadministration is subcutaneous.

In one aspect, the disclosure provides compositions comprising a complexcontaining an antifolate and (a) cyclodextrin or (b) a cationictherapeutic agent. In some embodiments, the cationic therapeutic agentis a platinum-based drug.

Antifolates

Antifolates are a class of antiproliferative drugs that were designed tomimic folic acid in its systemic transport, physiologic cell uptake andintracellular processing. Monoglutamates are the only circulating formsof folates in the blood and the only form of folate that is transportedacross the cell membrane. Like monoglutamated folates, antifolates suchas MTX, PMX, LTX, AG2034, RTX, piritrexim, pralatrexate, AG2034, GW1843,aminopterin, and LY309887 are monoglutamated and are transported intocells by reduced folate carriers (RFCs) and membrane folate-bindingproteins, where they are polyglutamated, by the addition of ˜2-5 L-gammaglutamyl moieties. The L-gamma-polyglutamated forms of the antifolateare biologically active and inhibit enzymes involved in folatemetabolism. This inhibition of folate metabolism suppresses de novonucleotide biosynthesis, renders the cell incapable of undergoingaccurate DNA replication, and ultimately results in cell death.

In particular embodiments, the therapeutic agent is an antifolate or anantifolate-based chemotherapeutic agent.

As used herein, the term “antifolate” or “antifolate-basedchemotherapeutic agent” refers to a polyglutamated or non-polyglutamatedpolyglutamatable antifolate. In some embodiments, the polyglutamatableantifolate is a member selected from methotrexate (MTX), PMX, lometrexol(LTX), AG2034, raltitrexed (RTX), piritrexim, pralatrexate, AG2034,GW1843, aminopterin, and LY309887. In some embodiments, the antifolateis polyglutamated and is a member selected from the group:polyglutamated methotrexate (MTX), polyglutamated pemetrexed (PMX),polyglutamated lometrexol (LTX), polyglutamated AG2034, polyglutamatedraltitrexed (RTX), polyglutamated piritrexim, polyglutamatedpralatrexate, polyglutamated AG2034, polyglutamated GW1843,polyglutamated aminopterin, and polyglutamated LY309887.

Many antifolates contain one glutamyl group in their administered form.For example, pemetrexed(N-[4-2-(2-Amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-1-glutamicacid) contains one glutamyl group (monoglutamated). The addition ofglutamic acid residue(s) to pemetrexed results in polyglutamatedpemetrexed. For example, the addition of 5 more glutamic acid residuesto pemetrexed would lead to a total of 6 glutamyl groups (one frompemetrexed and 5 additional glutamyl groups added) and is referred to inthis document as hexaglutamated pemetrexed or pemetrexed hexaglutamate.In the literature this may also be referred to as pentaglutamatedpemetrexed or pemetrexed pentaglutamated.

As used herein, the term “antifolate-based liposome composition” refersa liposome composition that contains a complex of an antifolate-basedtherapeutic agent and a cyclodextrin, and/or a liposome compositionsthat contains a complex of an antifolate-based chemotherapeutic agentand a cyclodextrin and that also contains a complex of a platinum-basedchemotherapeutic agent and a cyclodextrin. These liposome compositionsprovide improvements to the efficacy and safety of deliveringantifolate-based agents to cancer cells by providing the preferentialdelivery of a more cytotoxic payload (e.g., polyglutamated antifolates)compared to the cytotoxicity of the corresponding antifolatae-basedagent delivered in its uncomplexed and non-liposome encapsulated state.

In one aspect, the disclosure provides compositions comprising a complexcontaining an antifolate and (a) cyclodextrin or (b) a cationictherapeutic agent. In some embodiments, the cationic therapeutic agentis a platinum-based drug. In some embodiments, theantifolate-platinum-based drug complex comprises cisplatin or a saltthereof. In some embodiments, the platinum-based drug complex comprisesoxaliplatin, or a salt thereof.

In one embodiment, the disclosure provides a liposome compositioncomprising a complex of an antifolate-based therapeutic agent and acyclodextrin; one or more pharmaceutically acceptable carriers; and apegylated liposome. In some embodiments, the antifolate-basedtherapeutic agent is a member selected from MTX, PMX, LTX, AG2034, RTX,piritrexim, pralatrexate, AG2034, GW1843, aminopterin, and LY309887. Insome embodiments, the antifolate-based therapeutic agent ispolyglutamated and is a member selected from the group: polyglutamatedmethotrexate (MTX), polyglutamated pemetrexed (PMX), polyglutamatedlometrexol (LTX), polyglutamated AG2034, polyglutamated raltitrexed(RTX), polyglutamated piritrexim, polyglutamated pralatrexate,polyglutamated AG2034, polyglutamated GW1843, polyglutamatedaminopterin, and polyglutamated LY309887.

In some embodiments, the antifolate-based therapeutic agent ispemetrexed. In further embodiments, the antifolate-based therapeuticagent is polyglutamated pemetrexed. In further embodiments, theantifolate-based therapeutic agent is polyglutamated pemetrexedcontaining a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 glutamylgroups. In some embodiments, the polyglutamated pemetrexed contains atotal of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 L-gamma peptidelinkages. In other embodiments, the polyglutamated pemetrexed contains atotal of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 D-gamma peptidelinkages. In additional embodiments, the polyglutamated pemetrexedcontains a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 alphapeptide linkages.

In some embodiments, the antifolate-based therapeutic agent ismethotrexate. In further embodiments, the antifolate-based therapeuticagent is polyglutamated methotrexate. In further embodiments, theantifolate-based therapeutic agent is polyglutamated methotrexatecontaining a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 glutamylgroups. In some embodiments, the polyglutamated methotrexate contains atotal of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 L-gamma peptidelinkages. In other embodiments, the polyglutamated methotrexate containsa total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 D-gamma peptidelinkages. In additional embodiments, the polyglutamated methotrexatecontains a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 alphapeptide linkages.

In some embodiments, the antifolate-based therapeutic agent islometrexol. In further embodiments, the antifolate-based therapeuticagent is polyglutamated lometrexol. In some embodiments, theantifolate-based therapeutic agent is polyglutamated lometrexolcontaining a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 glutamylgroups. In some embodiments, the polyglutamated lometrexol contains atotal of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 L-gamma peptidelinkages. In other embodiments, the polyglutamated lometrexol contains atotal of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 D-gamma peptidelinkages. In additional embodiments, the polyglutamated lometrexolcontains a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 alphapeptide linkages.

In some embodiments, the antifolate-based therapeutic agent ispralatrexate. In some embodiments, the antifolate-based therapeuticagent is polyglutamated pralatrexate. In some embodiments, theantifolate-based therapeutic agent is polyglutamated pralatrexatecontaining a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 glutamylgroups. In some embodiments, the polyglutamated pralatrexate contains atotal of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 L-gamma peptidelinkages. In other embodiments, the polyglutamated pralatrexate containsa total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 D-gamma peptidelinkages. In additional embodiments, the polyglutamated pralatrexatecontains a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10 alphapeptide linkages.

In some embodiments, the antifolate-based therapeutic agent is apolyglutamated antifolate selected from the group: polyglutamatedAG2034, polyglutamated raltitrexed (RTX), polyglutamated piritrexim,polyglutamated AG2034, polyglutamated GW1843, polyglutamatedaminopterin, and polyglutamated LY309887. In some embodiments, thepolyglutamated antifolate contains a total of 2, 3, 4, 5, 6, 7, 8, 9,10, 2-6 or 2-10 glutamyl groups. In some embodiments, the polyglutamatedantifolate contains a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10L-gamma peptide linkages. In other embodiments, the polyglutamatedantifolate contains a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10D-gamma peptide linkages. In additional embodiments, polyglutamatedantifolate contains a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10alpha peptide linkages.

In some embodiments, the antifolate contained in an antifolate-basedliposome composition disclosed herein is an antifolate as depicted inFIG. 1. In some embodiments, that antifolate is an antifolate asdepicted in FIG. 1. Additional gamma L polyglutamated antifolates thatcan be included within the anti-folate-based liposome compositionsprovided herein are disclosed in International Application No.PCT/US2017/046667, the contents of which is herein incorporated byreference in its entirety.

In some embodiments, the antifolate contained in an antifolate-basedliposome composition disclosed herein is an antifolate as depicted inFIG. 2. Additional alpha- and gamma D polyglutamated antifolates thatcan be included within the anti-folate-based liposome compositionsprovided herein are disclosed in International Application No.PCT/US2017/046666, the contents of which is herein incorporated byreference in its entirety.

In a distinct aspect, the disclosure provides liposome compositions thatcontain liposomes encapsulating a complex of a anti-folate and acyclodextrin such as a derivatized beta cyclodextrin. In someembodiments, antifolate is polyglutamated (e.g., a polyglutamatedantifolate having the structure Antifolate-(α-L-glutamyl)n, where n=2,3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10). In some embodiments, thepolyglutamate is selected from the group: MTX, PMX, LTX, AG2034, RTX,piritrexim, pralatrexate, AG2034, GW1843, aminopterin, and LY309887.

Accordingly, in one embodiment, the disclosure provides a liposomecomposition comprising liposomes encapsulating a complex of anantifolate and a cyclodextrin. In some embodiments, the liposomes arepegylated. In some embodiments, the antifolate is polyglutamated (e.g.,a polyglutamate having the structure Antifolate-(α-L-glutamyl)n, wheren=2, 3, 4, 5, 6, 7, 8, 9, 10, 2-6 or 2-10). In some embodiments, thepolyglutamate is selected from the group: MTX, PMX, LTX, AG2034, RTX,piritrexim, pralatrexate, AG2034, GW1843, aminopterin, and LY309887.

In some embodiments, the cyclodextrin complexed with theantifolate-based therapeutic agent in the provided liposome compositionsis derivatized or underivatized. In some embodiments, the cyclodextrinis derivatized. In further embodiments, the cyclodextrin is aderivatized beta-cyclodextrin (e.g., a hydroxypropyl beta-cyclodextrin(HP-beta-CD), and a sulfobutyl ether beta-CD (SBE)-beta-cyclodextrin).In some embodiments, the cyclodextrin is a derivatized beta-cyclodextrincomprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2-hydroxylpropyl-3-group substitutions of hydroxy groups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more sulfoalkyl ether group substitutions of hydroxy groups. Infurther embodiments, the cyclodextrin is a derivatized beta-cyclodextrincomprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfobutyl ethergroup substitutions of hydroxy groups.

In some embodiments, the cyclodextrin complexed with theantifolate-based therapeutic agent is a derivatized cyclodextrin ofFormula I:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branched C₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group.

In some embodiments, the cyclodextrin complexed with theantifolate-based therapeutic agent is a derivatized cyclodextrin ofFormula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺², and ammonium ions andamine cations such as the cations of (C1-C6)-alkylamines, piperidine,pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine.

In some embodiments, the liposomes in the liposome composition comprisebetween 100 to 100,000 antifolate-based therapeutic agent/cyclodextrincomplexes. In additional embodiments, the liposome composition comprisesliposomes that have a diameter in the range of 20 nm to 200 nm. In someembodiments, liposomes in the composition comprise between 100 to100,000 platinum. In some embodiments, liposomes in the composition areanionic or neutral. In further embodiments, liposomes in the compositionhave a zeta potential that is less than or equal to zero. In furtherembodiments, liposomes in the composition has a zeta potential that isbetween 0 to −150 mV. In other embodiments, liposomes in the compositionare cationic. In further embodiments, liposomes in the composition havea zeta potential that is between 1 and 100 mV.

The liposomes in the provided antifolate-based liposome compositions arepreferably pegylated. In some embodiments, the polyethylene glycol ofthe liposomes has a number average molecular weight (Mn) of 200 to 5000daltons. The internal phase of the liposome has a pH in the range of2.8-6.8. In some embodiments, the internal phase of the liposomecomprises trehalose (e.g., 5% to 20% weight of trehalose).

In additional embodiments, the antifolate-based liposome compositioncomprises liposomes that contain a targeting moiety. In someembodiments, the targeting moiety is attached to one or both of a PEGand the exterior of the liposome. In additional embodiments, thetargeting moiety has a specific affinity for a surface antigen on atarget cell of interest. In some embodiments, the targeting moiety is apolypeptide. In additional embodiments, the targeting moiety is anantibody, a humanized antibody, an antigen binding fragment of anantibody, a single chain antibody, a single-domain antibody, abi-specific antibody, a synthetic antibody, a pegylated antibody, or amultimeric antibody. In some embodiments, the liposome compositioncomprises liposomes that contain from 30 to 500 targeting moieties. Infurther embodiments, the liposome composition comprises liposomes thatcontain from 30 to 200 targeting moieties.

In some embodiments, the antifolate-based liposome composition comprisesliposomes that further contain one or more of an immunostimulatoryagent, a detectable marker and a maleimide disposed on at least one ofthe PEG and the exterior of the liposome.

In some embodiments, the disclosed antifolate-based liposomecompositions further comprise a second complex formed by a therapeuticagent or a salt thereof, and a cyclodextrin. In some embodiments, thetherapeutic agent of the second complex is a member selected from thegroup: gemcitabine, and doxorubicin (including salts and free formsthereof). The cyclodextrin of the second complex in thegemcitabine-based liposome compositions can be derivatized orunderivatized. In some embodiments, the cyclodextrin is derivatized. Infurther embodiments, the cyclodextrin is a derivatized beta-cyclodextrin(e.g., a hydroxypropyl beta-cyclodextrin (HP-beta-CD), and a sulfobutylether beta-CD (SBE)-beta-cyclodextrin). In some embodiments, thecyclodextrin of the second complex in the gemcitabine-based liposomecomposition is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more 2-hydroxylpropyl-3- group substitutions ofhydroxy groups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkylether group substitutions of hydroxy groups. In further embodiments, thecyclodextrin of the second complex is a derivatized beta-cyclodextrincomprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfobutyl ethergroup substitutions of hydroxy groups.

In some embodiments, the cyclodextrin of the second complex contained inthe liposomes of the antifolate-based liposome composition (i.e., thecomplex formed by a therapeutic agent (including a salt and free formthereof), and a cyclodextrin) is a derivatized cyclodextrin of FormulaI:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branched C₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3- group

In some embodiments, the cyclodextrin of the second complex contained inthe liposomes of the antifolate-based liposome composition is aderivatized cyclodextrin of Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺², and ammonium ions andamine cations such as the cations of (C₁-C₆)-alkylamines, piperidine,pyrazine, (C₁-C₆)-alkanolamine and (C₄-C₈)-cycloalkanolamine.

In some embodiments, the liposomes in the antifolate-based liposomecomposition comprise between 100 to 100,000 of the second complex formedby the therapeutic agent, and the cyclodextrin. In additionalembodiments, the liposome composition comprises liposomes that have adiameter in the range of 20 nm to 200 nm. In some embodiments, liposomesin the composition comprise between 100 to 100,000 antifolate-basedagent/cyclodextrin complexes. In some embodiments, the cyclodextrin ofthe second complex is different from the cyclodextrin of theantifolate-based agent complex. In some embodiments, the liposomecomprises the same cyclodextrin in the second complex and the antifolateagent-based complex. In some embodiments, the liposome comprises acyclodextrin in the second complex that is different from thecyclodextrin of the antifolate-based agent complex.

According to some embodiments, the liposomes of the antifolate-basedliposome compositions comprise oxidized phospholipids. In furtherembodiments, the phospholipids that are oxidized are a member selectedfrom the group consisting of phosphatidylserines, phosphatidylinositols,phosphatidylethanolamines, phosphatidylcholines and1-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate.

In additional embodiments, the antifolate-based liposome compositionscomprise oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylchloine (OxPAPC). Infurther embodiments, the oxPAPCs are epoxyisoprostane-containingphospholipids. In further embodiments, the oxPAPC is1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine(5,6-PEIPC),1-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine(PECPC) and/or 1-palmitoyl-2-(epoxy-isoprostaneE2)-sn-glycero-4-phosphocholine (PEIPC).

According to some embodiments, the provided antifolate-based liposomecompositions further comprise one or more of an immunostimulatory agent,a detectable marker and a maleimide, wherein the immunostimulatoryagent, the detectable marker or the maleimide is attached to the PEG orthe exterior of the liposome. In some embodiments, the immunostimulatingagent is at least one is a member selected from the group: a proteinimmunostimulating agent; a nucleic acid immunostimulating agent; achemical immunostimulating agent; a hapten; and an adjuvant. In someembodiments, the immunostimulating agent is at least one selected fromthe group: a fluorescein; a fluorescein isothiocyanate (FITC); a DNP; abeta glucan; a beta-1,3-glucan; and a beta-1,6-glucan. In someembodiments, the immunostimulatory agent and the detectable marker isthe same. In some embodiments, the liposome composition comprises ahapten. In further embodiments, the hapten comprises one or more offluorescein or Beta 1,6-glucan.

In some embodiments, the liposomes of the disclosed antifolate-basedliposome compositions further comprises at least one cryoprotectantselected from the group consisting of mannitol; trehalose; sorbitol; andsucrose. In additional embodiments, the provided liposomal compositionis in unit dosage form. In some embodiments, pharmaceutical compositionscomprising the liposome compositions disclosed herein are provided.

In some embodiments, the disclosure is directed to the use of thedisclosed antifolate-based liposome compositions in the treatment ofdisease. In some embodiments, the disclosure is directed to use of theliposome compositions in the manufacture of a medicament for thetreatment of disease.

In some embodiments, the disclosure provides a method of killing ahyperproliferative cell comprising contacting a hyperproliferative cellwith a disclosed antifolate-based liposome composition. In furtherembodiments, the hyperproliferative cell is a cancer cell.

In some embodiments, the disclosure provides a method for treating orpreventing disease in a subject needing such treatment or prevention,the method comprising administering an effective amount of aantifolate-based liposome composition provided herein to a subject inneed thereof. In some embodiments, the administration is parenteral. Insome embodiments, the administration is intravenous. In someembodiments, the administration is subcutaneous

In additional embodiments, the disclosure provides a method for treatingcancer in a subject, comprising administering an effective amount of aantifolate-based liposome composition disclosed herein to a subjecthaving or at risk of having cancer. In further embodiments, the canceris a member selected from the group: lung cancer, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, head and neck cancer,gastric cancer, gastrointestinal cancer, colorectal cancer, esophagealcancer, cervical cancer, liver cancer, kidney cancer, biliary ductcancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldyscrasias. In some embodiments, the administration is parenteral. Insome embodiments, the administration is intravenous. In someembodiments, the administration is subcutaneous

In other embodiments, the disclosure provides a method for treatingcancer in a subject, comprising administering an effective amount of aantifolate-based liposome composition to a subject, wherein the liposomecomprises a targeting moiety with specific affinity for an antigenexpressed on the surface of a cancer cell, and wherein the subject hasor is at risk of having a cancer cell that expresses the antigen. Infurther embodiments, the antigen is a folate receptor. In someembodiments, the administration is parenteral. In some embodiments, theadministration is intravenous. In some embodiments, the administrationis subcutaneous.

A method for treating cancer that comprises administering an effectiveamount of an antifolate-based liposome composition provided herein to asubject, wherein the liposome comprises a targeting moiety with specificaffinity for an antigen expressed on the surface of a cancer cell, andwherein the subject has or is at risk of having a cancer cell thatexpresses the antigen. In some embodiments, the administration isparenteral. In some embodiments, the administration is intravenous. Insome embodiments, the administration is subcutaneous

Also provided is maintenance therapy that comprises administering aneffective amount of a liposome composition disclosed herein to a subjectthat is undergoing or has undergone cancer therapy. In some embodiments,the administration is parenteral. In some embodiments, theadministration is intravenous. In some embodiments, the administrationis subcutaneous.

Formulations.

In some embodiments, the liposome composition is provided as apharmaceutical composition containing the liposome and a carrier, e.g.,a pharmaceutically acceptable carrier. Examples of pharmaceuticallyacceptable carriers contained in the provided pharmaceuticalcompositions include normal saline, isotonic dextrose, isotonic sucrose,Ringer's solution, and Hanks' solution. In some embodiments, a buffersubstance is added to maintain an optimal pH for storage stability ofthe pharmaceutical composition. In some embodiments, the pH of thepharmaceutical composition is between 6.0 and 7.5. In some embodiments,the pH is between 6.3 and 7.0. In further embodiments, the pH is 6.5.Ideally the pH of the pharmaceutical composition allows for bothstability of liposome membrane lipids and retention of the entrappedentities. Histidine, hydroxyethylpiperazine-ethylsulfonate (HEPES),morpholipoethylsulfonate (MES), succinate, tartrate, and citrate,typically at 2-20 mM concentration, are exemplary buffer substances.Other suitable carriers include, e.g., water, buffered aqueous solution,0.4% NaCl, and 0.3% glycine. Protein, carbohydrate, or polymericstabilizers and tonicity adjusters can be added, e.g., gelatin, albumin,dextran, or polyvinylpyrrolidone. The tonicity of the composition can beadjusted to the physiological level of 0.25-0.35 mol/kg with glucose ora more inert compound such as lactose, sucrose, mannitol, or dextrin.These compositions can routinely be sterilized using conventional,sterilization techniques known in the art (e.g., by filtration). Theresulting aqueous solutions can be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous medium prior to administration.

The provided liposome compositions can also contain otherpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, and tonicity adjusting agents, for example, sodium acetate,sodium lactate, sodium chloride, potassium chloride, and calciumchloride. Additionally, the liposome suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alpha-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The liposome concentration in the provided fluid pharmaceuticalformulations can vary widely depending upon need, e.g., from less thanabout 0.05% usually or at least about 2-10% to as much as 30 to 50% byweight and will be selected primarily by fluid volumes, and viscosities,in accordance with the particular mode of administration selected. Forexample, the concentration may be increased to lower the fluid loadassociated with treatment. This may be particularly desirable inpatients having atherosclerosis-associated congestive heart failure orsevere hypertension. Alternatively, liposome pharmaceutical compositionscomposed of irritating lipids may be diluted to low concentrations tolessen inflammation at the site of administration.

Some embodiments relate to a method of delivering a targeted pegylatedliposome containing complexes of a platinum-based chemotherapeutic agentand one or more polyglutamate molecules. An exemplary method comprisesthe step of administering a liposome composition comprising a targetedpegylated liposome containing complexes of a platinum-basedchemotherapeutic agent and one or more polyglutamate molecules (e.g., acomposition as described herein) in an amount sufficient to deliver aneffective dose of the platinum-based chemotherapeutic agent to thetumor.

Some embodiments relate to a method of delivering a targeted pegylatedliposome containing complexes of a platinum-based chemotherapeutic agentand a cyclodextrin. An exemplary method comprises the step ofadministering a liposome composition comprising a targeted pegylatedliposome containing complexes of a platinum-based chemotherapeutic agentand a cyclodextrin (e.g., a composition as described herein) in anamount sufficient to deliver an effective dose of the platinum-basedchemotherapeutic agent to the tumor.

The amount of liposome composition administered will depend upon theparticular cyclodextrin or polyglutamate complexed therapeutic agentencapsulated inside the liposomes, the disease state being treated, thetype of liposomes being used, and the judgment of the clinician.Generally the amount of liposome pharmaceutical composition administeredwill be sufficient to deliver an effective dose of the particulartherapeutic agent.

The quantity of liposome composition necessary to deliver an effectivedose can be determined by routine in vitro and in vivo methods, commonin the art of drug testing. See, for example, D. B. Budman, A. H.Calvert, E. K. Rowinsky (editors). Handbook of Anticancer DrugDevelopment, L W W, 2003. Effective dosages for various therapeuticcompositions are known to those skilled in the art. In some embodiments,a therapeutic entity delivered via the pharmaceutical liposomecomposition and provides at least the same or higher activity than theactivity obtained by administering the same amount of the therapeuticentity in its routine non-liposome formulation. Typically the dosagesfor the liposome pharmaceutical composition is in a range for example,between about 0.005 and about 500 mg of the therapeutic entity perkilogram of body weight, most often, between about 0.1 and about 100 mgtherapeutic entity/kg of body weight.

Pharmaceutical compositions comprising the provided liposomecompositions are also provided. Pharmaceutical compositions are sterilecompositions that comprise a sample liposome and preferably aplatinum-based chemotherapeutic agent, cyclodextrin, polyglutamate,platinum-polyglutamate complex, and/or platinum-cyclodextrin complex,preferably in a pharmaceutically-acceptable carrier.

Unless otherwise stated herein, a variety of administration routes areavailable. The particular mode selected will depend, upon the particularactive agent selected, the particular condition being treated and thedosage required for therapeutic efficacy. The provided methods can bepracticed using any known mode of administration that is medicallyacceptable and in accordance with good medical practice. In someembodiments, the administration route is an injection. In furtherembodiments, the injection is by a parenteral route elected from anintramuscular, subcutaneous, intravenous, intraarterial,intraperitoneal, intraarticular, intraepidural, intrathecal,intravenous, intramuscular, or intra sternal injection. In someembodiments, the administration route is an infusion. In additionalembodiments, the administration route is oral, nasal, mucosal,sublingual, intratracheal, ophthalmic, rectal, vaginal, ocular, topical,transdermal, pulmonary, or inhalation.

In some embodiment, the liposomes are prepared as an infusioncomposition, an injection composition, a parenteral composition, or atopical composition. In further embodiments, the injection includes oneor more of: intraperitoneal injection, direct intratumor injection,intra-arterial injection, and intravenous injection, subcutaneousinjection, intramuscular injection, delivery via transcutaneous andintranasal route. In a further embodiment, the liposome composition is aliquid solution or a suspension. However, solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection arealso provided herein. In some embodiments, the liposome composition isformulated as an enteric-coated tablet or gel capsule according tomethods known in the art.

In some embodiments, the liposome compositions are administered to atumor of the central nervous system using a slow, sustained intracranialinfusion of the liposomes directly into the tumor (e.g., aconvection-enhanced delivery (CED)). See, Saito et al., Cancer Research64:2572-2579 (2004); Mamot et al., J. Neuro-Oncology 68:1-9 (2004). Inother embodiments, the liposome compositions are directly applied totissue surfaces. Sustained release, pH dependent release, and otherspecific chemical or environmental condition-mediated releaseadministration of the components of the liposome (e.g., depot injectionsand erodible implants) are also provided. Examples of suchrelease-mediating compositions are further provided herein or otherwiseknown in the art.

For administration by inhalation, the compositions can be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,ichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount.

When it is desirable to deliver the compositions systemically, they canformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers. Pharmaceutical parenteral formulations include aqueoussolutions of the ingredients. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively,suspensions of liposomes can be prepared as oil-based suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides.

Alternatively, the liposome compositions can be in powder form orlyophilized form for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

The provided liposome compositions can also be formulated in rectal orvaginal compositions such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

The provided liposome compositions have applications, in vivo, ex vivoand in vitro. In some embodiments, the compositions have in vitroapplications. In vitro use may include uses such as cell culturing andtissue engineering where selective treatment of a subpopulation of cellsis desired. For example, during the culture of stem cells from a normalpatient or a patient suffering from cancer, the cells can be treatedwith a sample composition or sample liposome as discussed to addresscancerous subpopulations of cells. The cancerous subpopulation may arisebecause the donor originally has cancer or because the cellsspontaneously transform during in vitro procedures.

In some embodiments, the liposome compositions are provided in a kitcomprising a container with the liposomes, and optionally, a containerwith the entity (antigen) targeted or preferentially bound by liposomes,and an instruction, e.g., procedures or information related to using theliposome composition in one or more applications. Such instruction canbe provided via any medium, e.g., hard paper copy, electronic medium, oraccess to a database or website containing the instruction.

The embodiments, provide pharmaceutical compositions. Pharmaceuticalcompositions are sterile compositions that comprise a delivery vehiclesuch as a liposome encapsulating a platinum-cyclodextrin complex or aplatinum-polyglutamate complex, preferably in apharmaceutically-acceptable carrier.

When it is desirable to deliver the compositions systemically, they maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers. Pharmaceutical parenteral formulations include aqueoussolutions of the ingredients. Aqueous injection suspensions may containsubstances that increase the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol, or dextran. Alternatively,suspensions of liposomes may be prepared as oil-based suspensions.Suitable lipophilic solvents or vehicles include fatty oils, such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides.

Alternatively, the liposomal compositions may be in powder form orlyophilized form for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

Administration

Generally, the compositions of the present invention can be administeredto patients by various routes. The particular mode selected will depend,of course, upon the particular active agent selected, the particularcondition being treated and the dosage required for therapeuticefficacy. The provided methods, generally speaking, may be practicedusing any mode of administration that is medically acceptable, meaningany mode that produces effective levels of a desired response withoutcausing clinically unacceptable adverse effects. Administration routesfor the disclosed compositions include injections, by parenteral routes,such as subcutaneous, intravenous (including bolus injection),intramuscular, intraarterial, intraperitoneal, intraarticular,intraepidural, intrathecal, intravenous, intramuscular, intra sternalinjection or infusion, or others, as well as oral, nasal, mucosal,sublingual, intratracheal, ophthalmic, rectal, vaginal, ocular, topical,transdermal, pulmonary, and inhalation administration routes.

In one embodiment, the provided pharmaceutical liposome composition isprepared as an infusion composition, an injection composition, aparenteral composition, or a topical composition, either as a liquidsolution or suspension. However, solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. In an additional embodiment, the liposome composition isformulated into an enteric-coated tablet or gel capsule according tomethods know in the art.

For delivery of liposomal drugs formulated according to exampleembodiments, to tumors of the central nervous system, a slow, sustainedintracranial infusion of the liposomes directly into the tumor (aconvection-enhanced delivery, or CED) may be of particular advantage.See, Saito, et al., Cancer Research, (64): 2572-2579 (2004); Mamot etal., J. Neuro-Oncology (68):1-9 (2004). The compositions may, forexample, also be directly applied to tissue surfaces. Sustained release,pH dependent release, or other specific chemical or environmentalcondition mediated release administration is also specifically providedherein, e.g., by such means as depot injections, or erodible implants. Afew non limiting examples are listed below for illustration.

For oral administration, the compounds may readily formulated bycombining the disclosed liposomal compositions with pharmaceuticallyacceptable carriers known in the art. Such carriers enable formulationas for example, tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, films, and suspensions for oral ingestion by a subjectto be treated. Suitable excipients for include but are not limited tofillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations, such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). Optionally,the oral formulations are formulated in saline or buffers forneutralizing internal acid conditions or administered without carriers.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the liposomal composition suspended in suitableliquids, such as aqueous solutions, buffered solutions, fatty oils,liquid paraffin, or liquid polyethylene glycols. Stabilizers may also beadded. All formulations for oral administration should be in dosagessuitable for such administration.

For buccal administration, the compositions can take the form of tabletsor lozenges formulated in a conventional manner.

For administration by inhalation, the compositions may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,ichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount.

The compositions may also be formulated in rectal or vaginalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

The example embodiments, contemplate administration of agents tosubjects having or at risk of developing a cancer, including for examplea solid tumor cancer, using the compositions and liposomes of exampleembodiments.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparations ofthe disclosed compositions include without limitation aqueous vehicles,nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers,antioxidants, local anesthetics, suspending and dispersing agents,emulsifying agents, sequestering or chelating agents and otherpharmaceutically acceptable substances.

Examples of aqueous vehicles include sodium chloride, Ringers injection,isotonic dextrose, sterile water, dextrose and lactated Ringers.Examples of nonaqueous parenteral vehicles include fixed oils ofvegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.Antimicrobial agents in bacteriostatic or fungistatic concentrations arerequired to be added to parenteral preparations packaged inmultiple-dose containers. Such antimicrobial agents include phenols orcresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include, without limitation, phosphate and citrate.Examples of antioxidants include for example sodium bisulfate. Exemplarylocal anesthetics include for example procaine hydrochloride. Suspendingand dispersing agents include sodium carboxymethylcelluose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include for example, Polysorbate 80 (TWEEN® 80). A sequesteringor chelating agent of metal ions include EDTA. Pharmaceutical carriersalso include ethyl alcohol, polyethylene glycol and propylene glycol forwater miscible vehicles and sodium hydroxide, hydrochloric acid, citricacid or lactic acid for pH adjustment.

The concentration of a composition in an injectable formulation providedherein can routinely be adjusted such that an injection provides aneffective amount to produce the desired pharmacological effect. Theexact dose depends on the age, weight and condition of the patient oranimal as is known or can routinely be determined by the skilledartisan.

The unit-dose parenteral preparations are packaged in an ampule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active ingredient is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In oneembodiment, an effective injectable dosage is formulated to contain aconcentration of at least about 0.1% w/w up to about 90% w/w or more(preferably more than 1% w/w) of a compound provided herein to thetreated tissue(s). The active ingredient may be administered at once, ormay be divided into a number of smaller doses to be administered atintervals of time. It is understood that the precise dosage and durationof treatment is a function of the tissue being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is noted that the concentrationsand dosage values may also vary with the age of the individual treated.It is further noted that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the formulations, and that the concentrationranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed formulations.

The compositions provided herein may be suspended in micronized or othersuitable form or may be derivatized to produce a more soluble activeproduct or a prodrug. The form of the resulting mixture depends uponfactors that include for example, the intended mode of administrationand the solubility of a composition provided herein in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the condition and may be empiricallydetermined.

The compositions provided herein also includes lyophilized powders,which can be reconstituted for administration as solutions, emulsionsand other mixtures. The compositions can also be reconstituted andformulated as solids or gels. The lyophilized powder can be prepared forexample, by dissolving the active ingredient, or a pharmaceuticallyacceptable derivative thereof, in a suitable solvent. The solvent mayoptionally contain an excipient which improves the stability or otherpharmacological property of the powder or reconstituted solution,prepared from the powder. Excipients that may be used include, but arenot limited to, dextrose, sorbital, fructose, corn syrup, xylitol,glycerin, glucose, sucrose or other suitable agent. The solvent may alsocontain a buffer, such as citrate, sodium or potassium phosphate orother such buffer known to those in the art, for maintain a desired pH(e.g., about neutral pH). Subsequent sterile filtration of the solutionfollowed by lyophilization under standard conditions known in the artcan routinely be applied to obtain the desired formulation. Generally,the resulting solution is apportioned into vials for lyophilization. Insome embodiments, each vial contains a single dosage (10-500 mg,preferably 100-300 mg) or multiple dosages of a composition providedherein. The lyophilized powder can be stored under appropriateconditions know in the art (e.g., about 4° C. to room temperature).Reconstitution of the lyophilized powder with water for injectionprovides an exemplary, non-limiting formulation for use in parenteraladministration. For reconstitution, about 1-50 mg, preferably 5-35 mg,more preferably about 9-30 mg of lyophilized powder, is added per mL ofsterile water or other suitable carrier. The precise amount depends uponthe selected compound. Such amount can be empirically determined usingtechniques known in the art.

EXAMPLES Example 1: Liposomal Alpha Polyglutamated PemetrexedCompositions

Methods:

Production of Polyglutamate—Cisplatin Complexes

To produce polyglutamate—Diammine dicarboxylic acid platinum (DDAP)Complex), an alpha hexaglutamate and Diammine dicarboxylic acid platinum(DDAP) was used. The process of complexation is dependent on thepresence of Chlorinated platinum compound and pH conditions. Thecomplexation is achieved by a nucleophilic attack on one or two carboxylgroups of glutamate by the platinate derivative. Briefly the complex wasformed by the following procedure. First, the active compound DDAP wasweighed and dissolved in 5% dextrose. After the DDAP dissolution step,aG6 is weighed out and added to the DDAP solution and allowed to stirfor 1 hour at 37° C. The pH of the solution is adjusted to 6.5-7.0 using1N NaOH and the solution was stirred overnight at 37° C. The formationof complex was confirmed visually. However when the pH was adjusted toacidic pH (3-4). The color reverted to its original. —The formation oflight brown color indicated the formation of the complex. Thisobservation was confirmed using HPLC Data not shown.

Production of Pentaglutamated Pemetrexed-DDAP Complex (PGPD) Liposomes

Briefly PGPD is encapsulated in liposomes by the following procedure.First, the lipid components of the liposome membrane are weighed out andcombined as a concentrated solution in ethanol at a temperature ofaround 65° C. In this example, the lipids used are hydrogenated soyphosphatidylcholine, cholesterol, and DSPE-PEG-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]).The molar ratio of HSPC:Cholesterol:PEG-DSPE is approximately 3:2:0.15.Next, PGPD is prepared as described in claim 0001 The PGPD drug solutionis heated up to 65° C. The ethanolic lipid solution is injected into thePGPD solution using a small-bore needle. During this step the drugsolution is well stirred using a magnetic stirrer. The mixing isperformed at an elevated temperature (63° C.-72° C.) to ensure that thelipids are in the liquid crystalline state (as opposed to the gel statethat they attain at temperatures below the lipid transition temperatureTm=51° C.−54° C.). As a result, the lipids are hydrated and formmultiple bilayer (multilamellar) vesicles (MLV) containing PGPD in theaqueous core.

Downsizing of MLV's Using Filter Extrusion

The MLVs are fragmented into unilamellar (single bilayer) vesicles ofthe desired size by high-pressure extrusion using two passes throughstacked (track-etched polycarbonate) membranes. The stacked membraneshave two layers with a pore size of 200 nm and six layers with a poresize of 100 nm. During extrusion, the temperature is maintained abovethe Tm to ensure plasticity of the lipid membranes. Because of theextrusion, large and heterogeneous in size and lamellarity MLVs turninto small, homogenous (100-120 nm) unilamellar vesicles (ULV) thatsequester the drug in their interior. A Malvern Zetasizer Nano ZSinstrument (Southborough, Mass.) with back scattering detector (90°) wasused for measuring the hydrodynamic size (diameter) at 25° C. in aquartz micro cuvette. The samples were diluted 50-fold in formulationmatrix before analysis.

Purification of Liposomes

After the ULV's containing PGPD have been produced, the extra-liposomalPGPD is removed using columns for small volume or tangential flowdiafiltration against a suitable buffer for large volume. Although anybuffer solution can be used, in this example the buffer used is 5 mMHEPES, 145 mM Sodium Chloride, pH 6.7. Upon completion of purification,filter sterilization is performed using a 0.22-micron filter

Production of Alpha Hexaglutamated Pemetrexed (αHgPMX) Liposomes

The foregoing description of the specific embodiments, will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

This application claims priority to U.S. Appl. No. 62/583,432, filedNov. 8, 2017, the contents of which are herein incorporated by referencein its entirety. The breadth and scope of the present invention shouldnot be limited by any of the above-described exemplary embodiments, butshould be defined only in accordance with the following claims and theirequivalents.

1. A liposome composition comprising a liposome encapsulating (a) acomplex of a platinum-based chemotherapeutic agent or a salt thereof andone or more polyglutamates or (b) a complex of a platinum-basedchemotherapeutic agent or a salt thereof and a cyclodextrin; and one ormore pharmaceutically acceptable carriers; and a pegylated liposome. 2.The liposome composition of claim 1, wherein the platinum-basedchemotherapeutic agent is cisplatin or a cisplatin analog, or whereinthe platinum-based chemotherapeutic agent is a member selected from thegroup: cisplatin, oxaliplatin, stratoplatin, paraplatin, platinol,cycloplatin, dexormaplatin, spiroplatin picoplatin, nedaplatin,triplatin, tetraplatin, lipoplatin, lobaplatin, ormaplatin, zeniplatin,platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK 121, CI-973,DWA 2114R, NDDP, and dedaplatin.
 3. (canceled)
 4. The liposomecomposition of claim 1, wherein the liposome encapsulates a complex of aplatinum-based chemotherapeutic agent or a salt thereof and acyclodextrin, and wherein the cyclodextrin is a derivatized orunderivatized beta-cyclodextrin.
 5. The liposome composition of claim 4,wherein the cyclodextrin is a derivatized beta-cyclodextrin, optionallywherein the cyclodextrin (CD) is a hydroxypropyl beta-CD (HP-beta-CD),or a sulfobutyl ether beta-CD ((SBE)-beta-CD); or the cyclodextrin is aderivatized beta-cyclodextrin comprising: (a) 1, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more 2-hydroxylpropyl-3- group substitutions of hydroxygroups; or (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkyl ethergroup substitutions of hydroxy groups; or the cyclodextrin is aderivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more sulfobutyl ether group substitutions of hydroxy groups. 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. The liposome composition ofclaim 5, wherein: the cyclodextrin is a derivatized beta-cyclodextrin ofFormula III:

wherein R equals: (a) (H)_(21-X) or (—(CH₂)₄—SO₃Na)_(x), and x=1.0-10.0,1.0-5.0, 6.0-7.0 or 8.0-10.0; (b) (H)_(21-X) or (—(CH₂CH(OH)CH₃)_(x),and x=1.0-10.0, 1.0-5.0, 6.0-7.0 or 8.0-10.0; (c) (H)_(21-X) or(sulfoalkyl ether)_(x), and x=1.0-10.0, 1.0-5.0, 6.0-7.0 or 8.0-10.0; or(d) (H)_(21-X) or (—(CH₂)₄—SO₃Na)_(x), and x=1.0-10.0, 1.0-5.0, 6.0-7.0or 8.0-10.0; the cyclodextrin is a derivatized cyclodextrin of FormulaI:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branched C₁-C₈-alkylene group, a 2-hydroxylpropyl-3- group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈- alkylene group or a 2-hydroxylpropyl-3- group; or cyclodextrin isa derivatized cyclodextrin of Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)—SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation.
 10. (canceled)
 11. (canceled)
 12. The liposome composition ofclaim 9, wherein the pharmaceutically acceptable cation is selectedfrom: an alkali metal such as Li⁺, Na⁺, or K⁺; an alkaline earth metalsuch as Ca⁺², or Mg+²; and ammonium ions and amine cations such as thecations of (C₁-C₆)-alkylamines, piperidine, pyrazine,(C₁-C₆)-alkanolamine, and (C₄-C₈)-cycloalkanolamine.
 13. The liposomecomposition of claim 1, wherein each liposome comprises between 100 to100,000 complexes formed by the platinum-based chemotherapeutic agent orsalt thereof.
 14. The liposome composition of claim 1, wherein theliposome has a diameter in the range of 20 nm to 200 nm or in the rangeof 80 nm to 120 nm.
 15. (canceled)
 16. The liposome composition of claim1, wherein the polyethylene glycol of the liposome has a number averagemolecular weight (Mn) of 200 to 5000 daltons.
 17. (canceled)
 18. Theliposome composition of claim 1, which comprises a steric stabilizerselected from the group consisting of polyethylene glycol (PEG);poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone)(PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline);poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol;poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilicpoly-N-vinylpyrrolidones; L-amino-acid-based polymer; and polyvinylalcohol.
 19. The liposome composition of claim 1, wherein the liposomecomprises at least one of an anionic lipid and a neutral lipid, orwherein the liposome comprises at least one is a member selected fromthe group: DSPE; DSPE-PEG-maleimide; DSPE-PEG-FITC; HSPC; HSPC-PEG;cholesterol; cholesterol-PEG; and cholesterol-maleimide.
 20. (canceled)21. The liposome composition of claim 1, wherein the liposome comprisesoxidized phospholipids, optionally wherein the phospholipids are amember selected from the group consisting of phosphatidylserines,phosphatidylinositols, phosphatidyl-ethanolamines, phosphatidylcholinesand 1-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate.
 22. The liposomecomposition of claim 1, wherein the liposome comprises oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylchloine (OxPAPC),optionally wherein the oxPAPCs are epoxyisoprostane-containingphospholipids, or wherein the oxPAPC is1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine(5,6-PEIPC),1-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine(PECPC) and/or 1-palmitoyl-2-(epoxy-isoprostaneE2)-sn-glycero-4-phosphocholine (PEIPC).
 23. (canceled)
 24. The liposomecomposition of claim 1, wherein the liposome is anionic or neutral, orwherein the liposome is cationic.
 25. The liposome composition of claim1, wherein the liposome has a zeta potential that is: (a) less than orequal to zero, (b) between 0 to −150 mV or between −30 to −50 mV, (c)greater than zero, or (d) between 1 and 100 mV, between 5 to 60 mV, orbetween 10 to 50 mV.
 26. (canceled)
 27. The liposome composition ofclaim 1, wherein the pH of the internal phase of the liposome is between2.5 and 7.5.
 28. (canceled)
 29. The liposome composition of claim 1,wherein the liposome internal phase comprises trehalose, optionallywherein the liposome comprises 5% to 20% weight of trehalose.
 30. Theliposome composition of claim 1, which further comprises a targetingmoiety attached to one or both of a PEG and the exterior of theliposome, and wherein the targeting moiety has a specific affinity for asurface antigen on a target cell of interest, optionally wherein thetargeting moiety is attached to one or both of the PEG and the exteriorof the liposome by a covalent bond herein.
 31. (canceled)
 32. Theliposome composition of claim 30, wherein the targeting moiety is apolypeptide, optionally, wherein the targeting moiety is an antibody ora fragment of an antibody.
 33. The liposome composition of claim 30,wherein the targeting moiety binds the surface antigen with anequilibrium dissociation constant (Kd) in a range of 0.5×10⁻¹⁰ to10×10⁻⁶ as determined using BIACORE® analysis.
 34. The liposomecomposition of claim 30, wherein the targeting moiety specifically bindsone or more folate receptors selected from the group: folate receptoralpha (FR-α), folate receptor beta (FR-β), and folate receptor delta(FR-δ).
 35. The liposome composition of claim 30, wherein the targetingmoiety comprises one or more members selected from the group: anantibody, a humanized antibody, an antigen binding fragment of anantibody, a single chain antibody, a single-domain antibody, abi-specific antibody, a synthetic antibody, a pegylated antibody, and amultimeric antibody.
 36. The liposome composition of claim 30 whereineach PEGylated liposome comprises from 30 to 500 targeting moieties or30 to 200 targeting moieties.
 37. The liposome composition of claim 30,wherein the liposome does not comprise a targeting moiety attached toone or both of a PEG and the exterior of the liposome.
 38. (canceled)39. The liposome composition of claim 1, wherein the liposome furthercomprises a second complex formed by a therapeutic agent or a saltthereof, and one or more polylglutamate molecules or a cyclodextrin,optionally, the therapeutic agent of the second complex is gemcitabineor doxorubicin, or a salt thereof.
 40. (canceled)
 41. The liposomecomposition of claim 1, further comprising one or more of animmunostimulatory agent, a detectable marker and a maleimide, whereinthe immunostimulatory agent, the detectable marker or the maleimide isattached to the PEG or the exterior of the liposome, optionally whereinthe immunostimulating agent is at least one member selected from thegroup: a protein immunostimulating agent; a nucleic acidimmunostimulating agent; a chemical immunostimulating agent; a hapten;and an adjuvant, or at least one selected from the group: a fluorescein;a fluorescein isothiocyanate (FITC); a DNP; a beta glucan; abeta-1,3-glucan; and a beta-1,6-glucan
 42. (canceled)
 43. The liposomecomposition of claim 1, which further comprises at least onecryoprotectant selected from the group consisting of mannitol;trehalose; sorbitol; and sucrose.
 44. A method of killing ahyperproliferative cell comprising contacting a hyperproliferative cellwith the liposome composition of claim 1, optionally wherein thehyperproliferative cell is a cancer cell.
 45. A method for treating orpreventing disease in a subject needing such treatment or prevention,the method comprising administering an effective amount of the liposomecomposition of claim 1 to a subject in need thereof, optionally whereinthe disease is cancer and the subject has or at risk of having cancer.46. The method of claim 45, wherein the cancer is a member selected fromthe group: lung cancer, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma; and a hematologicmalignancy such as for example, a leukemia, a lymphoma and other B cellmalignancies, myeloma and other plasma cell dyscrasias.
 47. A method ofdelivering a liposome composition to a tumor expressing an antigen onits surface, the method comprising: administering the liposomecomposition of claim 30 to a subject having a tumor expressing theantigen bound by the liposome targeting moiety in an amount to deliver atherapeutically effective dose of the liposome composition to the tumor,optionally wherein the administration is parenteral or intravenous. 48.A method for treating cancer that comprises administering an effectiveamount of the liposome composition of claim 30 to a subject having or atrisk of having a cancer cell that expresses on its surface the antigenbound by the liposome targeting moiety, optionally wherein theadministration is parenteral or intravenous.
 49. A maintenance therapythat comprises administering an effective amount of the liposomecomposition of claim 1 to a subject that is undergoing or has undergonecancer therapy, optionally wherein the administration is parenteral orintravenous.
 50. A pharmaceutical composition comprising the liposomecomposition of claim 1.